Phosphonate functional antimicrobial coatings for metal surfaces

ABSTRACT

The invention relates to quaternary ammonium multi-dentate mono-, bis-, tris- and tetrakis-phosphonate compounds, processes for preparing quaternary ammonium multi-dentate mono-, bis-, tris- and tetrakis-phosphonate compounds, antimicrobial coating compositions comprising quaternary ammonium multi-dentate mono-, bis-, tris- and tetrakis-phosphonate compounds and method of treating a surface with said compositions to provide a durable, antimicrobial-treated surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/CA2014/050796 filedAug. 20, 2014, which claims benefit of PCT/CA2014/000104 filed Feb. 12,2014, which claims benefit of U.S. Provisional Application 61/766,533filed Feb. 19, 2013, all of which are incorporated herein by referencein their entireties. This application is also a continuation-in-part ofPCT/CA2014/000104 filed Feb. 12, 2014, which claims benefit of U.S.Provisional Application 61/766,533 filed Feb. 19, 2013, all of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Bacterial infections in hospital environments are spread by twodifferent ways: external contamination or in vivo contamination fromimplants. Patients can develop external infections through contact withsurfaces such as door handles, pens, telephones, health care workersuniforms (“HCWU”), stethoscopes, or sterile packaging that have beencolonized by microorganisms. Hospital-acquired infections (“HAI”) fromcontact with pathogenic microorganisms affect approximately 2 millionpeople and result in more than 100,000 deaths in the U.S.A. each year.Such infections require 10-20 days of additional patienthospitalization, costing the already strained U.S. health-care systemsapproximately $25,000-30,000 per infection totaling billions of dollarsper year.

The second route for bacteria to infect patients is through hospitalinvasive support equipment such as intravascular lines and implantedmedical devices such as artificial prosthetics, cardiovascular implantsand urinary catheters. Implant associated infections (“IAI”) occur inmore than one million patients and cost an estimated $3 billion in theU.S. per year. For example, approximately 10-50% of patients withimplanted catheters run the risk of developing urinary tract infections(“UTI”) resulting in additional healthcare costs. The rise in thefrequency and severity of HAI's and IAI's can be attributed to decreasedantibiotic efficacy against drug-resistant strains of pathogens found insurface biofilms.

Biofilm formation involves three phases beginning with the initialreversible adhesion of bacteria on a surface through polysaccharides andadhesion proteins on the bacterial membrane (phase I). Under appropriateconditions, bacteria subsequently firmly attach to a surface (phase II),followed by the secretion of a protective polymeric matrix (biofilm,phase III) in which the bacteria typically show a marked increase inresistance to antibiotics, compared to none-adherent bacteria. As aresult, once the infection occurs, it becomes difficult to treat. Thus,strategies that prevent bacterial contamination or destroy adsorbedmicroorganisms that lead to biofilm formation are actively sought.

Listeria has been observed to increase biofilm production whenconditions become more unstable or non-ideal for active growth (Nilsson,R., Ross, T. and Bowman, J. (2011) Variability in biofilm production byListeria monocytogenes correlated to strain origin and growthconditions. International Journal of Food Microbiology 150. Pages14-24). It appears that Listeria use biofilm production as a form ofdefense, and the strength of their survival seems linked to the maturityof the biofilm as does their resistance to antimicrobials.

Biofilms allows for essential cell to cell interactions as well asproviding protection to harmful conditions. Listeria biofilms arestructurally simple and a mature community can be formed after 24 hours,which is the incubation period of the large droplet tests.

In general, actively growing Listeria cells are susceptible toquaternary ammonium compounds (QACs), even at relative short exposuretimes over broad temperature and pH ranges. In contrast, it appears thatmature biofilms are more resistant against QACs, which suggests that acomponent of the mature biofilm, potentially an extra-cellular polymericsubstance (EPS), could provide protection against QACs. This behaviorcould be unique to Listeria strains, but there are likely othermicroorganisms that that possess similar mechanisms for survivingexposure to antimicrobials.

In addition to biofilm maturity, it appears that other factors such ashigh pH and smooth attachment surfaces may impact on antimicrobialefficacy of QACs against Listeria (Yang, H., Kendall, P., Medeiros andL., Sofos, J. (2009) Efficacy of sanitizing agents against Listeriamonocytogenes biofilms on high-density polyethylene cutting boardsurfaces. Journal of Food Protection, Vol. 72, No. 5, Pages 990-998).Lastly the possibility of non-lethal doses of disinfectants beingexposed to the community over time results in resistance by themicrobial community (Feliciano, L., Li, J., Lee, J., Pascall, M. (2012)Efficacies of sodium hypochlorite and quaternary ammonium sanitizers forreduction of norovirus and selected bacteria during ware-washingoperations. PLOS ONE, Vol 7, Issue 12. E50273). The Listeria strainsisolated could be of a stock that has built up these resistances.

In order to prevent the formation of biofilm, strategies have beenemployed in the past to make surfaces inhospitable to bacteria. Forexample, small molecule monolayers or polymer thin films either “graftedto” or “grown from” a surface have been widely used to prepareantimicrobial surfaces and clothing. These prior art monolayers orpolymer coatings include, for example, non-biofouling coatings which arepassive strategies that rely on preventing bacterial adhesion withhydrophobic or zwitterionic thin films, but do not kill the approachingbacteria. A second class of antibacterial thin films kills microbes oncontact either by releasing a biocidal agent or immobilizing a biocidalagent. A third class of antibacterial thin films utilize a combinationstrategy of including a non-biofouling and biocidal component into thecoating.

Organophosphorus Antimicrobial Surfaces Based on Monolayers

The first quaternary ammonium phosphonate compounds (phosphonate quats)were disclosed in the early 1950's in U.S. Pat. No. 2,774,786 and Dutchpatent NL 79189 for use as synthetic detergents. In the patentssyntheses, the final product could only be isolated as a sodium salt ofthe phosphobetaine after hydrolysis of the phosphonate ester with HClfollowed by treatment with NaHCO₃. In a similar synthesis Germanaud etal., (Bulletin de la Societe Chimique de France, 1988, 4, 699-704)published the isolation of the phosphonate quats as betaines bypurification on an anion exchange resin. The products disclosed in thepatents were not spectrally characterized and were used as is, whileGermanaud's purification was costly and the product wasn't isolated as aphosphonic acid.

Phosphonate monolayers for the antimicrobial treatment of surfaces havebeen shown to be advantageous over self-assembled monolayers (SAMs) ofthiols and silanes in terms of durability, long-term stability andsurface coverage, especially on titanium and stainless steel.Thiol-based SAM's lack substrate specificity (mainly reserved for goldsurfaces) and long-term stability needed for biomedical applications,(i.e. implants). Over time, the thiol-based SAM's become oxidized tosulfonates, which lack affinity for gold and become displaced from thesurface.

In comparison to silane based SAM's on metal oxide surfaces, phosphonatebased SAM's are advantageous because they resist hydrolysis underphysiological conditions and higher surface coverage can be obtainedwithout harsh acid surface pretreatment (to increase the OH content).Siloxanes are also known to be unstable and are easily hydrolyzed underphysiological conditions.

Both active and passive strategies to prevent biofilm formation havebeen described with both mono- and bis-phosphonate monolayers. Examplesfor active surfaces include contact killing monolayers employingimmobilized quaternary ammonium salts and the antibiotic daptomycin.Passive strategies have been described employing hydrophobicperfluorinated bisphosphonates on stainless steel, silicon, and titaniumoxidize surfaces for anticorrosion applications.

U.S. Pat. No. 4,101,654 teaches phosphonate-pendant nitrogenheterocyclic compounds that are quaternized by alkyl halides and theiruse as corrosion inhibitor compounds.

U.S. Pat. No. 4,420,399 teaches phosphonate-quaternary ammoniumcompounds having a methylene group linking the phosphorus and nitrogenatoms and their use as corrosion inhibitor compounds.

U.S. Pat. No. 4,962,073 teaches porous surfaces treated with phosphoricacid esters.

U.S. Pat. No. 5,770,586 teaches phosphonate/phosphoric acid-quaternaryammonium compounds for use as dental care ingredients and for bonedensity treatment.

U.S. Pat. No. 5,888,405 teaches methods of inhibiting bacteria fromadhering to submerged surfaces using amino-phosphoric acid compounds.

U.S. Patent Application Publication No. 2002/0023573 teachesphosphonate, phosphate and phosphinate compounds linked to mineral oxidesurfaces through the oxygen atoms of the phosphorus moieties.

U.S. Patent Application Publication No. 2002/0128150 teachesphosphonate, phosphate and phosphinate sulfur compounds linked tomineral oxide surfaces through the oxygen atoms of the phosphorusmoieties.

PCT Application Publication WO 2007/080291 teaches bisphosphonate-aminesand quaternary ammonium compounds, their preparation and attachment tometal and metal-oxide surfaces and testing for antibacterial activity.

PCT Application Publication WO 2008/017721 teaches bisphosphonate-aminesand quaternary ammonium compounds, their preparation and attachment tosilicon and metal surfaces and cell proliferation testing.

U.S. Patent Application Publication No. 2008/0220037 teachesbisphosphonic acid compounds having pendant oxygen, sulfur or at leasttwo quaternary ammonium functional groups, their preparation andtreatment of mineral and metal surfaces and antibacterial or biofilmformation testing.

Guerro G et al., Pathologie Biologie, 2009, 57, 36-43 teaches surfacesmodified with materials such as phosphonate quaternary ammoniumcompounds and phosphonate silver coatings, and their bacterial adhesionand inhibition properties.

Queffelec C et al., Chemical Reviews, 2012, 112(7), 3777-3807 teachesphosphonic acids and esters, their synthesis and modification ofsurfaces using functionalized phosphonic acids and esters. Thefunctional groups include heterocycles, amino groups and larger organicmolecules.

Thus, there has been a long-felt need for a durable and environmentallysafe antimicrobial metal or mineral surface treatment and a process tomanufacture the same, which is more effective against a variety ofmicrobes than existing antimicrobial surface treatments.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided a quaternaryammonium mono-phosphonate compound of formula (I) and a process forpreparing a compound of formula (I)

wherein R₁ and R₂ are independently lower alkyl groups preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, more preferably selected from methyl, ethyl, isopropyl orn-propyl groups, most preferably methyl groups, in is 15, 16, 17, 18 or19, most preferably 17, n is 0, 1, 2, 3, 4, 5 or 6, most preferably 1,and X is chloro, bromo or iodo, most preferably bromo, comprising thesteps of(a) reacting a compound of formula (II)

where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, most preferably ethyl or isopropyl, with an alkylhalide of formula (III)

where X and n are as above and Y is a halogen selected from chloro orbromo, most preferably bromo to give a compound of formula (IV)

(b) reacting the compound of formula (IV) with a compound of formula (V)

wherein R₁ and R₂ are each independently a lower alkyl group, preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, more preferably selected from methyl, ethyl, isopropyl orn-propyl groups, most preferably methyl groups, and m is 15, 16, 17, 18or 19, most preferably 17, to give a compound of formula (VI)

and (c) reacting a compound of formula (VI) with SiR₃R₄R₅Z wherein R₃,R₄ and R₅ are independently methyl or ethyl and Z is chloro, bromo, iodoor triflate, or a mineral acid selected from HCl, HBr or HI, to give acompound of formula (I). In a preferred embodiment the process may takeplace neat or in a polar, protic reaction solvent, preferably a loweralkanol selected from methanol, ethanol and isopropanol. The process maybe carried out at the refluxing temperature of the reaction solvent. Theprocess is considered complete when the compound of formula (VI) is nolonger observable via thin-layer chromatography. Optionally the compoundof formula (I) may be purified, preferably by chromatography orrecrystallization.

According to another aspect of the invention there is provided aquaternary ammonium bis-phosphonate compound of formula (VII) and aprocess for preparing a compound of formula (VII)

wherein R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butylor phenyl, preferably the same, more preferably ethyl, and Z is chloro,bromo, hydroxyl, or iodo, preferably bromo, R₁ and R₂ are eachindependently a lower alkyl group, preferably saturated hydrocarbonchains being one, two or three carbon atoms in length, more preferablymethyl, m is 15, 16, 17, 18 or 19, n is 0, 1, 2, 3, 4, 5, or 6, and o is1, 2 or 3, comprising the steps of(a) reacting, preferably at least two equivalents of compound of formula(IX)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, per equivalent of a compoundof formula (X)

to give a compound of formula (XI)

which is then reacted with a compound of formula (V)

where R, R₁, R₂, m, n and Z are as defined above, to give a compound offormula (VII).

According to another aspect of the invention there is provided aquaternary ammonium bis-phosphonate compound of formula (VII) and aprocess for preparing a compound of formula (VII) wherein R′ isindependently hydrogen, methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl or hydrogen and even morepreferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, more preferably methyl groups, m is 15, 16, 17,18 or 19 and Z is chloro, bromo or hydroxyl, preferably bromo,comprising the steps of:

(a) reacting a compound of formula (XI)

where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, n is 0, 1, 2, 3, 4, 5, or 6,o is 1, 2 or 3 and Z is selected from chloro, bromo, hydroxyl or iodo,preferably bromo, with p-toluenesulfonyl chloride, trimethyl ammoniumchloride, trimethylamine in a polar, aprotic solvent preferablyacetonitrile, dimethylformamide or dichloromethane, more preferablydichloromethane,(b) adding a compound R₁R₂NH where R₁ and R₂ are each independently alower alkyl group, preferably saturated hydrocarbon chains being one,two or three carbon atoms in length, more preferably methyl, in a polar,protic solvent selected from methanol, ethanol or isopropanol optionallyin the presence of water, to give a compound of formula (XII)

and (c) reacting the compound of formula (XII) with a compound offormula (XIII)

where m is 15, 16, 17, 18 or 19, and Z is chloro, bromo hydroxyl oriodo, preferably bromo, to give a compound of formula (VII).

According to another aspect of the invention there is provided aquaternary ammonium bis-phosphonate of formula (XIV) and a process forpreparing a compound of formula (XIV)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, more preferably methyl groups, m is 15, 16, 17,18 or 19, n is 0, 1, 2, 3, 4, 5, or 6, and Z is selected from chloro,bromo or iodo, preferably bromo, comprising the steps of(a) reacting a compound of formula (XV) wherein R is defined as above

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, with a compound of formula(XVI)

to give a compound of formula (XVII)

which is treated with p-toluenesulfonic acid, methanesulfonyl chloride,triethylamine, and R₁R₂NH where R₁ and R₂ are defined as above, and acompound of formula (XIII)

where Z is chloro, bromo or iodo, preferably bromo to give a compound offormula (XIV),or (c) reacting a compound of formula (XVIII)

where n is as defined above with at least one equivalent of O═PH(OR)₂where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, in the presence of an alkalimetal carbonate, preferably potassium carbonate, methanesulfonylchloride and an organic amine base, and further reacted with sodiumhydride and at least a second equivalent of O═PH(OR)₂ to give a compoundof formula (XIX)

where R is as defined aboveand (d) reacting the compound of formula (XIX) with hydrazine, and analdehyde selected from formaldehyde or acetaldehyde in the presence ofzinc metal, and a compound of formula (XIII)

where Z is chloro or, bromo or iodo, preferably bromo, and m is 15, 16,17, 18 or 19, to give a compound of formula (XIV).

According to another aspect of the invention there is provided abis-phosphonate compound of formula (XVII) and a process for preparing acompound of formula (XVII)

where R is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl, and n is 0, 1, 2, 3,4, 5 or 6, comprising the steps of reacting a compound of formula (XX)

where R is as defined above, with a compound of formula (XXI)

to give a compound of formula (XVII).

According to another aspect of the invention there is provided abis-phosphonate compound of formula (XXII) and a process for preparing acompound of formula (XXII)

where R is methyl, ethyl, isopropyl, n-butyl or phenyl, preferably thesame, more preferably ethyl, R₁ and R₂ are each independently a loweralkyl group preferably saturated hydrocarbon chains being one, two orthree carbon atoms in length, more preferably methyl groups, and n is 0,1, 2, 3, 4, 5 or 6, comprising reacting a compound of formula (XXIII)

with O═P(OR)₂Cl where n and R are as defined above, in the presence oflithium diisopropylamide in a polar, aprotic solvent to give a compoundof formula (XXII) which optionally can be reacted with an alkyl halideof formula (XIII) to give a quaternary ammonium bis-phosphonate offormula (XIV) where R, R₁, R₂ and m are as defined above.

According to another aspect of the invention there is provided abis-phosphonate compound of formula (XXIV) and a process for preparing acompound of formula (XXIV) where R is hydrogen, methyl, ethyl,isopropyl, n-butyl or phenyl, preferably the same, more preferablyethyl, and n is 0, 1, 2, 3, 4, 5 or 6,

comprising reacting a compound of formula (XXV)

with O═P(OR)₂Cl where R and n are as defined above, in the presence oflithium diisopropylamide in a polar, aprotic solvent to give a compoundof formula (XXIV).

According to another aspect of the invention there is provided aquaternary ammonium bis-phosphonate compound of formula (XXVI) and aprocess for preparing a compound of formula (XXVI)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, more preferably methyl groups, m is 15, 16, 17,18 or 19 and Z is selected from chloro, bromo or iodo, preferably bromo,comprising the steps of(a) reacting oxalyl chloride with

in chilled dichloromethane in the presence of a compound of formula (II)to give a compound of formula (XXVII)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, and R₁ and R₂ are as definedabove, and(b) reacting the compound of formula (XXVII) with a compound of formula(XIII)

to give a compound of formula (XXVI) where R′, R₁, R₂, m and Z are asdefined above.

According to yet another aspect of the invention there is provided aquaternary ammonium mono-phosphonate compound of formula (XXVIII) and aprocess for preparing a compound of formula (XXVIII)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, more preferably methyl groups, n is 0, 1, 2, 3,4, 5 or 6, p is 0, 1, 2, 3, 4, 5 or 6, and Z is selected from chloro,bromo or iodo, preferably bromo, comprising the steps of(a) reacting a compound of (XXIX)

with a compound of formula (XXX)

where R₁, R₂ and p are as defined above, in a polar, aprotic solvent inthe presence of an organic amine base to give a compound of formula(XXXI)

and (b) reacting a compound of formula (XXXI) with a compound of formula(XXXII)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, and n and Z are as definedabove, in a polar, aprotic solvent to give a compound of formula(XXVIII).

In preferred embodiments, for chemical reactions involving reagents thatare sensitive to protons, the processes may take place neat or in polar,aprotic reaction solvents, for example but not limited todichloromethane, acetonitrile and dimethylformamide. For chemicalreactions involving reagents that are not sensitive to protons, theprocesses may take place in a lower alkanol preferably methanol, ethanoland isopropanol. The processes may be carried out at temperatures fromabout −80° C. to about 150° C. The process is considered complete whenthe starting material is no longer observable via thin-layerchromatography. The final products optionally may be purified,preferably by chromatography or recrystallization.

According to yet another aspect of the invention, there is providedquaternary ammonium multidentate tri- and tetra-substituted phosphonatecompounds of formula (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII) andprocesses for preparing the compounds of formula (XXXIII), (XXXIV),(XXXV), (XXXVI) and (XXXVII)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, m is 15, 16, 17, 18 or 19 and Z is selectedfrom chloro, bromo or iodo, preferably bromo, comprising the steps of:a) alkylating a tetralkyl methylenebisphosphonate (TAMBP) compound,mono-deprotecting TAMBP followed by mono alkylation to lead to alpha(C—H) bisphosphonates, and performing a second deprotonation/alkylationwith dialkyl chlorophosphate to provide trisphosphonates;b) Michael addition of dialkyl vinylphosphite to provide betaaminobisphosphonates and further deprotonation and phosphorylation withdialkyl chlorophosphate to provide tetraphosphonates; orc) Lewis acid-mediated Abrzov addition of trialkylphosphite three timesto three reactive bromoacteylTRISBOC and the radical addition of dialkylphosphite to terminal vinyl groups on the TRIS BOC scaffold to givetrisphosphonates.

According to yet another aspect of the invention there is provided anantimicrobial composition comprising any one of a compound of formulae(I), (VI), (VII), (XIV), (XXVI), (XXVIII), (XXXIII), (XXXIV), (XXXV),(XXXVI) and (XXXVII) and a process for treating a surface with anantimicrobial coating comprising the steps of contacting the surfacewith a composition comprising any one of a compound of formulae (I),(VI), (VII), (XIV), (XXVI), (XXVIII), (XXXIII), (XXXIV), (XXXV), (XXXVI)and (XXXVII).

According to yet another aspect of the invention, there is provided aphosphonate antimicrobial coating composition for treating surfaces togive a stable and durable phosphonate antimicrobial coating surfacetreatment, said composition comprising any one of a compound of formulae(I), (VI), (VII), (XIV), (XXVI), (XXVIII), (XXXIII), (XXXIV), (XXXV),(XXXVI) or (XXXVII) in a suitable carrier. In one embodiment saidsuitable carrier is an environmentally friendly carrier comprising alower alkanol selected from the group consisting of methanol, ethanol,n-propanol and i-propanol, water or a mixture thereof depending on thesolubility of the phosphonate compound in the carrier. The phosphonateantimicrobial coating can be applied onto a given surface preferably bydip coating, painting or with aerosol spraying with an about 1 to anabout 20 mM solution of the phosphonate compound for a length of time soas to completely coat the surface. In one embodiment, the coatingprocess may be repeated to apply additional layers of the phosphonateantimicrobial coating. Preferably the stable and durable phosphonateantimicrobial coatings may be coated onto various material surfaces suchas, but not limited to, metal oxides or metal alloys of aluminum,copper, iron, steel, titanium, zirconium and silicon (silica). Even morepreferably, phosphonate antimicrobial coating strength and stability maybe further enhanced by subjecting the uncoated surface to a pretreatmentoxidation step known as passivation (Min, S. L., Smiley, K. J. & Gawalt,E. S. J. Am. Chem. Soc. 193-204 (2011)). Without being bound by anytheory, passivation creates a metal hydroxide layer that providesadditional binding sites for the phosphonate compounds of thephosphonate antimicrobial coating to bind to. Passivation can beaccomplished known processes in the art such as thermal annealing(subjecting the uncoated surface to temperatures of about 100-140° C.for about 18 hours) or reduced pressure annealing (subjecting theuncoated surface to pressures of about 0.05 to about 0.3 Torr, morepreferably 0.1 Torr) (Raman, A., Dubey, M., Gouzman, I. & Gawalt, E. S.Formation of Self-Assembled Monolayers of Alkylphosphonic Acid on theNative Oxide Surface of SS316L. Langmuir 22, 6469-6472 (2006);Lecollinet, G. et al. Self-Assembled Monolayers of Bisphosphonates:Influence of Side Chain Steric Hindrance. Langmuir 25, 7828-7835(2009)).

According to yet another aspect of the invention, there is provided anantimicrobial surface coating comprising a phosphonate of compound offormulae (I), (VI), (VII), (XIV), (XXVI), (XXVIII), (XXXIII), (XXXIV),(XXXV), (XXXVI) or (XXXVII), said coating exhibiting activity against atleast one microbe. More preferably the compound of formula (I) isN-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammonium bromide and thecompounds of formula (VI) areN-(3-diethoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammonium bromideandN-(3-(diisopropoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide. In one embodiment, the surface may be a metal or a mineral,preferably a metal, preferably a metal oxide or metal alloy. Morepreferably the surface is selected from the group consisting ofaluminum, copper, iron, steel, titanium, zirconium and silica. Mostpreferably the surface is selected from aluminum, stainless steel ortitanium. Preferably the at least one microbe may be bacteria.Preferably the bacteria may be gram positive or gram negative bacteria.More preferably the bacteria is selected from the group consisting ofStaphylococcus, Pseudomonas, Klebsiella, Salmonella, Listeria,Arthrobacter and Escherichia. Most preferably the bacteria are selectedfrom the group consisting of Pseudomonas sp. CT07, Salmonellaenteriditis, Klebsiella pneumonia, Listeria monocytogenes, Arthrobacter,Staphylococcus aureus, Pseudomonas aeruginosa PA01 and Escherichia coli.Preferably the antimicrobial activity of the phosphonate antimicrobialcoating may be observed in less than about 24 hours, more preferably inless than about 6 hours and most preferably in less than about 4 hoursafter contact with bacteria. The antimicrobial surface coatingcomprising a phosphonate of formula (I), (VI), (VII), (XIV), (XXVI),(XXVIII), (XXXIII), (XXXIV), (XXXV), (XXXVI) or (XXXVII) havedemonstrated improved antimicrobial activity compared to existingantimicrobial surface treatments of the art.

According to yet another aspect of the invention, there is provided asurface coated with an antimicrobial surface coating as defined herein.

According to yet another aspect of the invention, there is provided amono-phosphonate compound of formula

According to yet another aspect of the invention, there is provided amono-phosphonate compound of formula

Further and other aspects will be appreciated by the skilled reader.

DETAILED DESCRIPTION OF THE INVENTION Brief Summary of Figures

FIG. 1 shows the ¹H NMR of compound (1) of Referential Example 1

FIG. 2 shows the ¹³C NMR of compound (1) of Referential Example 1

FIG. 3 shows the ³¹P NMR of compound (1) of Referential Example 1

FIG. 4 shows the ¹H NMR of compound (2) of Example 1

FIG. 5 shows the ¹³C NMR of compound (2) of Example 1

FIG. 6 shows the ³¹P NMR of compound (2) of Example 1

FIG. 7 shows the ¹H NMR of compound (3) of Example 2

FIG. 8 shows the ¹³C NMR of compound (3) of Example 2

FIG. 9 shows the ³¹P NMR of compound (3) of Example 2

FIG. 10 shows the ¹H NMR of compound (4) of Example 3

FIG. 11 shows the ¹³C NMR of compound (4) of Example 3

FIG. 12 shows the ³¹P NMR of compound (4) of Example 3

FIG. 13 shows the ¹H NMR of compound (5) of Example 4

FIG. 14 shows the ¹³C NMR of compound (5) of Example 4

FIG. 15 shows the ³¹P NMR of compound (5) of Example 4

FIG. 16 shows the ¹H NMR of compound (6) of Example 5

FIG. 17 shows the ³¹P NMR of compound (6) of Example 5

FIG. 18 shows the ¹H NMR of compound (7) of Example 6

FIG. 19 shows the ¹³C NMR of compound (7) of Example 6

FIG. 20 shows the ³¹P NMR of compound (7) of Example 6

FIG. 21 shows the ¹H NMR of compound (8) of Example 7

FIG. 22 shows the ¹³C NMR of compound (8) of Example 7

FIG. 23 shows the ³¹P NMR of compound (8) of Example 7

FIG. 24 shows the ¹H NMR of compound (9) of Example 8

FIG. 25 shows the ¹³C NMR of compound (9) of Example 8

FIG. 26 shows the ³¹P NMR of compound (9) of Example 8

FIG. 27 shows the ¹H NMR of compound (10) of Example 9

FIG. 28 shows the ¹³C NMR of compound (10) of Example 9

FIG. 29 shows the ¹H NMR of compound (11) of Example 10

FIG. 30 shows the ¹³C NMR of compound (11) of Example 10

FIG. 31 shows the ¹H NMR of compound (12) of Example 11

FIG. 32 shows the ³¹P NMR of compound (12) of Example 11

FIG. 33 shows the ¹H NMR of compound (13) of Example 12

FIG. 34 shows the ³¹P NMR of compound (13) of Example 12

FIG. 35 shows the ¹H NMR of compound (14) of Example 13

FIG. 36 shows the ¹³C NMR of compound (14) of Example 13

FIG. 37 shows the ³¹P NMR of compound (14) of Example 13

FIG. 38 shows the ¹H NMR of compound (15) of Example 14

FIG. 39 shows the ¹³C NMR of compound (15) of Example 14

FIG. 40 shows the ³¹P NMR of compound (15) of Example 14

FIG. 41 shows the ¹H NMR of compound (16) of Example 15

FIG. 42 shows the ¹³C NMR of compound (16) of Example 15

FIG. 43 shows the ³¹P NMR of compound (16) of Example 15

FIG. 44 shows the ¹H NMR of compound (17) of Example 16

FIG. 45 shows the ¹³C NMR of compound (17) of Example 16

FIG. 46 shows the ³¹P NMR of compound (17) of Example 16

FIG. 47 shows the ¹H NMR of compound (18) of Example 17

FIG. 48 shows the ¹³C NMR of compound (18) of Example 17

FIG. 49 shows the ¹H NMR of compound (19) of Example 18

FIG. 50 shows the ¹³C NMR of compound (19) of Example 18

FIG. 51 shows the ¹H NMR of compound (20) of Example 19

FIG. 52 shows the ¹³C NMR of compound (20) of Example 19

FIG. 53 shows the ³¹P NMR of compound (20) of Example 19

FIG. 54 shows the ¹H NMR of compound (21) of Example 20

FIG. 55 shows the ¹³C NMR of compound (21) of Example 20

FIG. 56 shows the ³¹P NMR of compound (21) of Example 20

FIG. 57 shows the ¹H NMR of compound (30) of Example 21

FIG. 58 shows the ¹³C NMR of compound (30) of Example 21

FIG. 59 shows the ¹H NMR of compound (31) of Example 22

FIG. 60 shows the ¹³C NMR of compound (31) of Example 22

FIG. 61 shows the ³¹P NMR of compound (31) of Example 22

FIG. 62 shows the ¹H NMR of compound (32) of Example 23

FIG. 63 shows the ¹³C NMR of compound (32) of Example 23

FIG. 64 shows the ³¹P NMR of compound (32) of Example 23

FIG. 65 shows a graph of the change in colony forming units (cfu) overtime upon exposure of untreated control (“C”), antimicrobial compound 2treated (“S1”), antimicrobial compound 3 treated (“S2”) and Bio-Protect®AM500 silicone quaternary ammonium salt treated (“BSC”) stainless steelcoupons to Pseudomonas sp. CT07, Salmonella enteriditis, Klebsiellapneumonia and Listeria monocytogenes.

FIG. 66—shows a graph of the change in colony forming units (cfu) overtime upon exposure of untreated control (“C”), antimicrobial compound 2treated (“S1”), antimicrobial compound 3 treated (“S2”) and Bio-Protect®AM500 silicone quaternary ammonium salt treated (“BSC”) stainless steelcoupons to Pseudomonas PA01, Arthrobacter, Staphylococcus aureus andEscherichia coli.

FIG. 67 shows a graph of the change in colony forming units over timeupon exposure of untreated control (“Ctr”) and antimicrobial compound 3treated titanium surfaces to Salmonella and S. aureus.

FIG. 68 shows single crystal x-ray structure of compound 67.

The present invention is directed to quaternary ammonium mono- andmultidentate-phosphonate compounds, methods for manufacturing thecompounds, compositions comprising said compounds and methods fortreating surfaces and/or articles with the compounds to provide adurable, antimicrobial-treated article.

The term quaternary ammonium mono-phosphonate refers to quaternaryammonium compounds that have been substituted with a single phosphonategroup O═P(OR)₂ where R is selected from methyl, ethyl, isopropyl,n-butyl or phenyl or hydrogen, preferably ethyl, isopropyl or hydrogenand even more preferably hydrogen, and the phosphonate group can belinked to the quaternary ammonium nitrogen centre by a one, two, threeor four carbon atom chain, preferably a saturated chain, more preferablya three carbon chain. The term quaternary ammoniummultidentate-phosphonate refers to quaternary ammonium compound thathave been substituted with two or more phosphonate groups O═P(OR)₂ whereR is as above.

The term polar, aprotic solvent means a solvent that has a dipole momentbut does not have an acidic hydrogen. Non-limiting examples includeacetonitrile, dimethylformamide, dimethylsulfoxide and dichloromethane.

The term polar, protic solvent means a solvent that has a dipole momentand has an acidic hydrogen. Non-limiting examples including loweralkanols, carboxylic acids and water.

The term surface means any metallic or non-metallic article surface thatis capable of forming phosphorus-oxygen bonds. Non-limiting examplesinclude steel, stainless steel, titanium, silica glass and clays.

The term neat means without the use of solvents, specifically directedto chemical reactions that do not involve the use of solvents.

All microwave reactions were performed in sealed glass reaction tubeutilizing the Biotage® Initiator Microwave Synthesizer at the indicatedtemperature and time.

The quaternary ammonium mono- and bis-phosphonate compounds of thepresent invention can be prepared via one of several processes. In oneembodiment, a quaternary ammonium mono-phosphonate compound of formula(I)

wherein R₁ and R₂ are each independently a lower alkyl group preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, most preferably methyl groups, m is 15, 16, 17, 18 or 19, mostpreferably 17, n is 0, 1, 2, 3, 4, 5 or 6, most preferably 1, and X ischloro, bromo or iodo, most preferably bromo, can be prepared by aprocess comprising the steps of(a) reacting a compound of formula (II)

where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same and most preferably ethyl or isopropyl, with analkyl halide of formula (III)

where n and X are as above and Y is a halogen selected from chloro orbromo, more preferably bromo to give a compound of formula (IV)

(b) reacting the compound of formula (IV) with a compound of formula (V)

wherein R₁ and R₂ are each independently a lower alkyl group, preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, most preferably methyl groups, and m is 15, 16, 17, 18 or 19,most preferably 17, to give a compound of formula (VI)

and (c) reacting a compound of formula (VI) with SiR₃R₄R₅Z wherein R₃,R₄ and R₅ are independently methyl or ethyl and Z is chloro, bromo, iodoor triflate, or a mineral acid selected from HCl, HBr or HI, to give acompound of formula (I). The process may take place neat or in a polar,protic reaction solvent, preferably a lower alkanol selected frommethanol, ethanol and isopropanol. The process may be carried out at therefluxing temperature of the reaction solvent. The process is consideredcomplete when the compound of formula (VI) is no longer observable viathin-layer chromatography. The final product optionally may be purified,preferably by chromatography or recrystallization. A particularlypreferred compound of formula (I) isN-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammonium bromide.Particularly preferred compounds of formula (VI) areN-(3-diethoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammonium bromideandN-(3-(diisopropoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide. A single crystal x-ray structure ofN-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammonium bromide is shownin FIG. 4.

In another embodiment, a quaternary ammonium bis-phosphonate compound offormula (VII)

wherein R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butylor phenyl, preferably the same, more preferably ethyl or hydrogen andeven more preferably hydrogen, R₁ and R₂ are each independently a loweralkyl group, preferably saturated hydrocarbon chains being one, two orthree carbon atoms in length, more preferably methyl groups, m is 15,16, 17, 18 or 19, n is 0, 1, 2, 3, 4, 5 or 6, o is 1, 2 or 3, and Z ischloro, bromo, hydroxy or iodo, preferably bromo, is prepared by aprocess comprising the steps of(a) reacting at least two equivalents of compound of formula (IX)

where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, with per equivalent of acompound of formula (X)

where n is 0, 1, 2, 3, 4, 5 or 6, and Z is selected from chloro, bromo,hydroxyl or iodo, preferably bromo, to give a compound of formula (XI)

where n, o, R and Z are as defined above, which is reacted with acompound of formula (V)

where R₁ and R₂ are independently lower alkyl groups preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, preferably methyl groups, and m is 15, 16, 17, 18 or 19, to givea compound of formula (VII) where R′, R₁, R₂, in, n, o and Z are asdefined above. The process for preparing the compound of formula (XI)can take place in a polar, aprotic solvent selected from but not limitedto acetonitrile or dichloromethane, or neat, preferably neat, at atemperature from about −5° C. to about 10° C. then warmed to about 90°C. to about 140° C. for about one hour. The product of formula (XI) canbe isolated by extraction and optionally purified, preferably bychromatography. The process alternatively can take place in the presenceof microwave radiation at a temperature of about 120° C. to about 140°C., preferably 130° C., for about five minutes. The microwave radiationhas a frequency of about 2500 MHz.

The process for preparing the compound of formula (VII) from thecompound of formula (XI) can take place in a neat mixture of a compoundof formula (XI) and a compound of formula (V) where R, R₁, R₂ and Z areas defined above. The process can take place in the absence of reactionsolvent at a temperature of about 90° C. to about 110° C., preferably100° C., for about one hour, or alternatively, in the presence ofmicrowave radiation at a temperature of about 140° C. to about 160° C.,preferably 150° C., for about one to three minutes, preferably about twominutes. The microwave radiation has a frequency of about 2500 MHz.

In an alternative embodiment, the quaternary ammonium bis-phosphonatecompound of formula (VII) is prepared by a process comprising the stepsof

(a) reacting a compound of formula (XI)

where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, R₁ and R₂ are eachindependently a lower alkyl group, preferably saturated hydrocarbonchains being one, two or three carbon atoms in length, more preferablymethyl groups, n is 0, 1, 2, 3, 4, 5 or 6, o is 1, 2 or 3, and Z isselected from chloro, bromo hydroxyl or iodo, preferably bromo, withp-toluenesulfonyl chloride, trimethyl ammonium chloride, an organicamine, preferably triethylamine, in a polar, aprotic solvent selectedfrom but not limited to acetonitrile, dimethylformamide ordichloromethane, preferably dichloromethane,(b) adding a compound R₁R₂NH where R₁ and R₂ are defined as above, in apolar, protic solvent selected from but not limited to methanol, ethanolor isopropanol, optionally in the presence of water, to give a compoundof formula (XII)

and (c) reacting the compound of formula (XII) with a compound offormula (XIII)

where m is 15, 16, 17, 18 or 19, and Z is chloro, bromo or, hydroxyl oriodo, preferably bromo, to give a compound of formula (VII). The processof step (a) can take place at a temperature of about 20° C. to about 30°C. The process of step (b) can take place at a temperature of about 90°C. to about 115° C., preferably 100° C., and for a reaction time ofabout one hour. Alternatively, the process of step (b) can take place inthe presence of microwave radiation at a temperature of about 100° C. toabout 120° C., preferably 110° C., for about five minutes. The microwaveradiation has a frequency of about 2500 MHz. The process of step (c) cantake place neat at a temperature of about 90° C. to about 115° C. forabout one hour or, alternatively, in the presence of microwave radiationat a temperature of about 140° C. to about 160° C., preferably 150° C.,for about two minutes. The microwave radiation has a frequency of about2500 MHz.

In another embodiment, the quaternary ammonium bis-phosphonate compoundof formula (XIV)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, more preferably methyl groups, m is 15, 16, 17,18 or 19, n is 0, 1, 2, 3, 4, 5 or 6, and Z is selected from chloro,bromo or iodo, preferably bromo, is prepared by a process comprising thesteps of(a) reacting a compound of formula (XV)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, with a compound of formula(XVI)

to give a compound of formula (XVII)

where n and R are as defined above, which is preferably not isolated andused in the next step, and(b) treating a compound of formula (XVII) with p-toluenesulfonic acid,methanesulfonyl chloride, triethylamine and R₁R₂NH where R₁ and R₂ aredefined as above, and a compound of formula (XIII)

where m is as defined above and Z is chloro, bromo or iodo, preferablybromo to give a compound of formula (XIV),or alternatively (c) reacting a compound of formula (XVIII)

wherein n is 0, 1, 2, 3, 4, 5, or 6, with one equivalent of O═PH(OR)₂where R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, in the presence of an alkalimetal carbonate, preferably potassium carbonate, methanesulfonylchloride and an organic amine base, preferably triethylamine, andfurther reacted with sodium hydride and a second equivalent of O═PH(OR)₂to give a compound of formula (XIX)

where n and R are as defined aboveand (d) reacting the compound of formula (XIX) with hydrazine, analdehyde selected from formaldehyde or acetaldehyde in the presence ofzinc metal, and a compound of formula (XIII)

where Z is chloro, bromo or iodo, preferably bromo, and m is 15, 16, 17,18 or 19, to give a compound of formula (XIV). The process of step (c)can take place in a polar, aprotic solvent selected from but not limitedto acetonitrile, tetrahydrofuran or dioxane, preferably acetonitrile ordioxane, at a temperature of about 25° C. to about 75° C., preferably60° C.

In another embodiment, the bis-phosphonate compound of formula (XVII)

where n is 0, 1, 2, 3, 4, 5, or 6, and R is independently methyl, ethyl,isopropyl, n-butyl or phenyl, preferably the same, more preferablyethyl, is prepared by a process comprising the step of reacting acompound of formula (XX)

where R is as defined above, with a compound of formula (XXI)

to give a compound of formula (XVII).

In another embodiment of preparing a compound of formula (XIV), thebis-phosphonate compound of formula (XXII)

where n is 0, 1, 2, 3, 4, 5 or 6, R is independently methyl, ethyl,isopropyl, n-butyl or phenyl, preferably the same, more preferablyethyl, R₁ and R₂ are each independently a lower alkyl group, preferablysaturated hydrocarbon chains being one, two or three carbon atoms inlength, more preferably methyl, is prepared by a process comprisingreacting a compound of formula (XXIII)

with O═P(OR)₂Cl where n, R, R₁ and R₂ are as defined above, in thepresence of lithium diisopropylamide in a polar, aprotic solvent to givea compound of formula (XXII) which optionally is reacted with an alkylhalide of formula (XIII) to give a quaternary ammonium bis-phosphonateof formula (XIV) where R, R₁, R₂, Z, m and n are as defined above.

In another embodiment, the compound of formula (XXIV)

where n is 0, 1, 2, 3, 4, 5 or 6, and R is independently methyl, ethyl,isopropyl, n-butyl or phenyl, preferably the same, more preferablyethyl, is prepared by a process comprising reacting a compound offormula (XXV)

with O═P(OR)₂Cl in the presence of lithium diisopropylamide in a polar,aprotic solvent to give a compound of formula (XXIV) where R isindependently methyl, ethyl, isopropyl, n-butyl or phenyl, preferablythe same, more preferably ethyl.

In another embodiment, the quaternary ammonium bis-phosphonate compoundof formula (XXVI)

where R′ is independently hydrogen, methyl, ethyl, isopropyl, n-butyl orphenyl, preferably the same, more preferably ethyl or hydrogen and evenmore preferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, preferably methyl groups, m is 15, 16, 17, 18 or19 and Z is selected from chloro, bromo or iodo, preferably bromo, isprepared by the process comprising the steps of(a) reacting oxalyl chloride with

in chilled dichloromethane in the presence of a compound of formula (II)to give a compound of formula (XXVII)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, and (b) reacting thecompound of formula (XXVII) with a compound of formula (XIII) to give acompound of formula (XXVI).

The process of step (a) can take place in a polar, aprotic solventselected from but not limited to acetonitrile or dichloromethane,preferably dichloromethane, and the term chilled means a temperaturefrom about −5° C. to about 10° C., rising to about 20° C. to about 30°C. for about one hour.

In another embodiment of the present invention, a quaternary ammoniummono-phosphonate compound of formula (XXVIII)

where n is 0, 1, 2, 3, 4, 5 or 6, p is 0, 1, 2, 3, 4, 5 or 6, R′ isindependently hydrogen, methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl or hydrogen and even morepreferably hydrogen, R₁ and R₂ are each independently a lower alkylgroup, preferably saturated hydrocarbon chains being one, two or threecarbon atoms in length, preferably methyl groups, m is 15, 16, 17, 18 or19 and Z is selected from chloro, bromo, iodo or mesyl, preferablybromo, is prepared by a process comprising the steps of(a) reacting a compound of (XXIX)

with a compound of formula (XXX)

where p, R₁ and R₂ are as defined above, in a polar, aprotic solvent inthe presence of an organic amine base to give a compound of formula(XXXI)

and (b) reacting a compound of formula (XXXI) with a compound of formula(XXXII)

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, and n and Z are as definedabove, in a polar, aprotic solvent to give a compound of formula(XXVIII). The dansyl group

is used as a UV fluorescing marker to indicate the presence of thequaternary ammonium mono-phosphate compound after a compound of formula(XXVIII) has been applied to a surface.

In another embodiment of the present invention, quaternary ammoniummultidentate tri- and tetra-substituted phosphonate compounds of formula(XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII)

are prepared where R′ is independently hydrogen, methyl, ethyl,isopropyl, n-butyl or phenyl, preferably the same, more preferably ethylor hydrogen and even more preferably hydrogen, R₁ and R₂ are eachindependently a lower alkyl group, preferably saturated hydrocarbonchains being one, two or three carbon atoms in length, preferably methylgroups, m is 15, 16, 17, 18 or 19 and Z is selected from chloro orbromo, preferably bromo. Multidentate phosphonic acid antimicrobials maygenerally be prepared by introduction of the bis-, tris- ortetraphosphonate anchor prior to quaternization and dealkylation withtrimethylbromosilane (TMBr). The most direct way to synthesizebisphosphonates is through the alkylation of tetralkylmethylenebisphosphonate (TAMBP). Monodeprotonation of TAMBP followed bymono alkylation leads to alpha (C—H) bisphosphonates whereas a seconddeprotonation/alkylation with dialkyl chlorophosphate providestrisphosphonates. A second method for the synthesis of bisphosphonatesis through Michael addition of dialkyl vinylphosphite to provide betaaminobisphosphonates. Further deprotonation and phosphorylation withdialkyl chlorophosphate provides tetraphosphonates. Alternatively theTRIS BOC scaffold containing three reactive groups may be turned intotrisphosphonates via established synthetic routes used to preparemonophosphonates. Two examples include the lewis acid-mediated Abrzovaddition of trialkylphosphite three times to three reactivebromoacteylTRISBOC and the radical addition of dialkyl phosphite toterminal vinyl groups on TRISBOC. General schemes for producingquaternary ammonium bis-, tris- and tetraphosphonate compounds are asfollows:

wherein R₁ and R₂ are independently lower alkyl groups, preferablymethyl.

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, R₁ and R₂ are independentlylower alkyl groups, preferably methyl and in is 15, 16, 17, 18 or 19.

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, R₁ and R₂ are independentlylower alkyl groups, preferably methyl and m is 15, 16, 17, 18 or 19.

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, R₁ and R₂ are independentlylower alkyl groups, preferably methyl and m is 15, 16, 17, 18 or 19.

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, and m is 15, 16, 17, 18 or19.

wherein R is independently methyl, ethyl, isopropyl, n-butyl or phenyl,preferably the same, more preferably ethyl, R₁ and R₂ are independentlylower alkyl groups, preferably methyl and m is 15, 16, 17, 18 or 19.

In another embodiment, the quaternary ammonium mono- and bis-phosphonateand multidentate tri- and tetra-substituted phosphonate compounds of thepresent invention may be used to antimicrobially treat hard surfaces. Asurface may be an inner surface and/or an outer surface. Specifically,there is provided a phosphonate antimicrobial coating composition fortreating surfaces to give a stable and durable phosphonate antimicrobialcoating surface treatment, said composition comprising any one of acompound of formulae (I), (VI), (VII), (XIV), (XXVI), (XXVIII),(XXXIII), (XXXIV), (XXXV), (XXXVI) or (XXXVII) in a suitable carrier.More preferably the compound of formula (I) isN-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammonium bromide and thecompounds of formula (VI) areN-(3-diethoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammonium bromideandN-(3-(diisopropoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide. In one embodiment said suitable carrier is an environmentallyfriendly carrier comprising a lower alkanol selected from the groupconsisting of methanol, ethanol, n-propanol and i-propanol, water or amixture thereof depending on the solubility of the phosphonate compoundin the carrier. The phosphonate antimicrobial coating may be appliedonto a given surface preferably by dip coating, painting or with aerosolspraying with an about 1 to an about 20 mM solution of the phosphonatecompound for a length of time so as to completely coat the surface. Inone embodiment, the coating process may be repeated to apply additionallayers of the phosphonate antimicrobial coating. Preferably the stableand durable phosphonate antimicrobial coatings may be coated ontovarious material surfaces such as, but not limited to, metal oxides ormetal alloys of aluminum, copper, iron, steel, titanium, zirconium andsilicon (silica). Even more preferably, phosphonate antimicrobialcoating strength and stability may be further enhanced by subjecting theuncoated surface to a pretreatment oxidation step known as passivation.Without being bound by any theory, passivation creates a metal hydroxidelayer that provides additional binding sites for the phosphonatecompounds of the phosphonate antimicrobial coating to bind to.Passivation is accomplished by known processes in the art such asthermal annealing (subjecting the uncoated surface to temperatures ofabout 100-140° C. for about 18 hours) or reduced pressure annealing(subjecting the uncoated surface to pressures of about 0.05 to about 0.3Torr, more preferably 0.1 Torr).

The hard surface coated with an antimicrobial coating comprising aphosphonate of compound of formulae (I), (VI), (VII), (XIV), (XXVI),(XXVIII), (XXXIII), (XXXIV), (XXXV), (XXXVI) or (XXXVII), particularlyN-(3-diethoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammonium bromide,N-(3-(diisopropoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide and N-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammoniumbromide, exhibits improved activity against at least one microbecompared to existing antimicrobial surface treatments, for example,Bio-Protect® AM500 silicone quaternary ammonium salt. Preferably the atleast one microbe may be bacteria. Preferably the bacteria may be grampositive or gram negative bacteria. More preferably the bacteria isselected from the group consisting of Staphylococcus, Pseudomonas,Klebsiella, Salmonella, Listeria, Arthrobacter and Escherichia. Mostpreferably the bacteria are selected from the group consisting ofPseudomonas sp. CT07, Salmonella enteriditis, Klebsiella pneumonia,Listeria monocytogenes, Arthrobacter, Staphylococcus aureus, Pseudomonasaeruginosa PA01 and Escherichia coli. Preferably the antimicrobialactivity of a phosphonate antimicrobial coating can be observed in lessthan about 24 hours, more preferably in less than about 6 hours and mostpreferably in less than about 4 hours after contact with bacteria.

With reference to FIG. 1, stainless steel coupons surface treated with acomposition comprising compound 2 (“S1”), compound 3 (“S2”) orcommercially-available Bio-Protect® AM500 silicone quaternary ammoniumsalt (“BSC”), and inoculated with Pseudomonas sp. CT07, Salmonellaenteriditis, Klebsiella pneumonia and Listeria monocytogenes. S1 and S2showed eradication of Pseudomonas sp. CT07, Salmonella enteriditis, andKlebsiella pneumonia in as little as four hours compared to BSC. AgainstListeria S1 and S2 showed comparable antibacterial activity compared toBSC. Listeria was indeed included in this study because it is widelyrecognized as a cause of listeriosis, an often-fatal foodborne disease.This organism is further known for its notorious persistence againstantimicrobial agents. The mechanisms for this resistance are notunderstood. Furthermore, these microorganisms are known to persist ondry surfaces over long periods of time. The result obtained, especiallywith S1 where it is completely eradicated after 24 hours, is thereforesignificant. It is also important to note that in the case of S2, therewas a reduction of more than 2 log (>100 times) compared to theuntreated control.

With reference to FIG. 2, stainless steel coupons surface treated with acomposition comprising compound 2 (“S1”), compound 3 (“S2”) orcommercially-available Bio-Protect® AM500 silicone quaternary ammoniumsalt (“BSC”), and inoculated with Pseudomonas PA01, Arthrobacter,Staphylococcus aureus and Escherichia coli. S1 and S2 showed eradicationof these strains in as little as four hours. Against Escherichia coli,S2 and S2 showed eradication in as little as four hours compared to BSCwhich took longer.

With reference to FIG. 3, titanium coupons surface treated with acomposition comprising compound 3 obtained by dealkylation of compound 2using trimethylsilyl bromide orN-(3-(diisopropoxylphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide using HCl showed eradication of Salmonella and S. aureus afterthree hours of drying on the titantium surface.

The following non-limiting examples are provided.

Acronyms:

AIBN—Azobisisobutyronitrile

ACN—acetonitrile

DCM—dichloromethane

DMF—dimethylformamide

Hrs—hours

LDA—lithiumdiisopropylamide

NaH—sodium hydride

MsCl—mesylchloride

ON—overnight

RT—room temperature

TMSBr—trimethylsilylbromide

TosCl—tosyl chloride

TOL—toluene

ST—sealed tube

uW—microwave

Monophosphonic Acid Quaternary Ammonium Antimicrobials (MPQ).

Referential Example 1 Diethyl (3-bromopropyl)phosphonate (1)

According to a general procedure reported in Li, F. et al.Photopolymerization of Self-Assembled Monolayers of DiacetylenicAlkylphosphonic Acids on Group-III Nitride Substrates. Langmuir 26,10725-10730 (2010), to a flame dried 250 mL round bottom flask equippedwith a reflux condenser connected to an inert atmosphere manifold, wasadded 1,3-dibromopropane (40 mL, 394 mmol, 4 eq.) followed bytriethylphosphite (13 mL, 75.8 mmol). The flask was evacuated (2 min),backfilled with N₂ and the reaction mixture refluxed overnight (175° C.)using a sand bath. The solution was then cooled to room temperature andexcess 1,3-dibromopropane was vacuum distilled (1×10⁻² mm Hg) using ashortpath distillation head attached to a Schlenk line. Once all of theexcess 1,3-dibromopropane was removed as judged by TLC, the titlecompound was vacuum distilled utilizing an oil bath (150° C.) to afforda clear, colourless liquid. Yield: 79% (15.54 g). TLC (50%EtOAc:hexanes, KMnO₄ stain), R_(f)=0.60; ¹H NMR (400 MHz, CDCl₃, δ):4.12-4.00 (m, 4H, H5), 3.43 (t, 2H, J=4.3 Hz, H4), 2.16-2.05 (m, 2H,H3), 1.90-1.81 (m, 2H, H2), 1.28 (t, J=7.0 Hz, 6H, H1) ppm; ¹³C NMR (100MHz, CDCl₃, δ): 61.6 (d, ²J_(C-P)=6.5 Hz, C5), 33.71 (C4), 25.92 (d,²J_(C-P)=4.4 Hz, C2), 23.64 (s, C3), 16.4 (d, ², J_(C-P)=6.2 Hz, C1)ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ): 30.2 ppm.

Example 1N-(3-diethoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammonium Bromide(2)

The compound has been previously reported in: Brunet, S., Germanaud, L.,Le, P., Pierre & Sillion, B. Neutral phosphobetaines, their preparation,and their use in petroleum recovery. Fr. Demande, 33 (1986); Chevalier,Y. et al. Zwitterionic amphiphiles: synthesis and physical properties.Commun. Journ. Corn. Esp. Deterg. 18, 231-45 (1987); Gallot, B.,Germanaud, L., Chevalier, Y. & Le, P., P. Mesomorphic structure ofneutral amphiphilic phosphotobetaines having different interionicdistances I. Ethylphosphonatobetaines. J. Colloid Interface Sci. 121,514-21 (1988); Germanaud, L., Brunel, S., Le, P., P. & Sillion, B.Surfactant properties of neutral phosphobetaines with a modulatedintercharge distance. Rev Inst Fr Pet 41, 773-85 (1986); and Germanaud,L., Brunel, S., Chevalier, Y. & Le, P., Pierre. Synthesis of neutralamphiphilic phosphobetaines with variable interionic distances. Bull.Soc. Chim. Fr., 699-704 (1988). To a flame dried and evacuated 20 mLscrew cap vial was added dimethyl (3-bromopropyl)phosphonate (1.264 g,4.88 mmol) followed by N,N-dimethyloctadecylamine (DMOA) by pasteurpipette (1.7075 g, 5.1 mmol, 1.1 eq.) and the closed vial placed in a100° C. sand bath for 35 min until it solidified. The mixture was thencooled to room temperature, centrifuged from hexanes (15 mL), andrecrystallized from 20 mL ethyl acetate/hexanes (1:5) to affordN-(3-(diethoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide as a white waxy solid. Yield: 67% (1.82 g). Mp=54-55° C.; ¹H NMR(400 MHz, CDCl₃, δ): 4.09-4.01 (m, 2H, H10), 3.66-3.22 (m, 2H, H9)3.43-3.38 (m, 2H, H8), 3.31 (s, 6H, H7), 2.03 (brs, 2H, H6), 1.84-1.80(m, 2H, H5), 1.67 (brs, 2H, H4), 1.33-1.25 (m, 6H, H3), 1.19 (brs, 30H,H2), 0.83-0.79 (m, 3H, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 64.45 (C8),63.09 (d, ³J_(C-P)=6.54 Hz C9), 62.97 (d, ²J_(C-P)=6.54 Hz, C10), 51.25(C7), 31.86 (C2 overlap), 29.64-29.19 (C2 overlap), 22.69 (C4), 22.62(C5), 16.45-16.39 (C6, C3), 14.05 (C1) ppm: ³¹P NMR (121.45 MHz, CDCl₃,δ): 29.54 ppm. HRMS-DART (m/z): [M⁺] calculated for C₃₃H₇₃N₂O₆P₂,476.4227. found, 476.4240.

Example 2 N-(3-phosphonopropyl)-N,N-dimethyloctadecan-1-ammonium Bromide(3)

The internal salt of this compound has been previously reported in:Martinelli, M. J. & Pollack, S. R. Bromotrimethylsilane, John Wiley &Sons Ltd, 2011); and Conibear, A. C., Lobb, K. A. & Kaye, P. T. 31P NMRkinetic study of the tandem cleavage of phosphonate esters bybromotrimethylsilane. Tetrahedron 66, 8446-8449 (2010). Inside a flamedried and evacuated 20 mL screw cap vialN-(3-(diethoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide (0.2768 g, 0.46 mmol) was dissolved in anhydrous DCM (5 mL). Tothe clear stirred solution was added TMSBr (0.25 mL, 1.9 mmol, 4.0 eq.)through a rubber septum via syringe and the reaction was stirred at roomtemperature overnight. Completion of the reaction was followed by ³¹Pafter which the reaction was quenched with EtOH (10 mL) and stirred for1 h followed by addition of H₂O (1 mL). Volatiles were removed with arotovap connected to a high vacuum Schlenk line and the crude productwas centrifuged with Et₂O (2×10 mL) to remove brown colored impurities(0.9422 g, 94%). A small portion of the title compound wasrecrystallized from EtOAc/IPA for MS & X-ray analysis. Clear, longneedles. Recrystallization of a sample of 3 from 80% EtOH/EtOAc producedx-ray quality crystals. The single crystal x-ray structure is shown inFIG. 4. Mp=118-120° C.; ¹H NMR (400 MHz, MeOD, δ): 3.38-3.33 (m, 2H,H8), 3.28-3.23 (m, 2H, H7), 3.02 (s, 6H, H6), 2.02-1.90 (m, 2H, H5),1.75-1.65 (m, 4H, H4, H3), 1.20 (brs, 30H, H3), 0.82 (t, J=6.9 Hz, 3H,H1), ppm; ¹³C NMR (CDCl₃, 100 MHz, δ): 64.26 (C7) 63.57 (C8), 49.94(C6), 31.68 (C2 overlap), 29.41-28.86 (C2 overlap), 26.03 (C2 overlap),22.45-22.17 (C3, C4), 16.47 (d, ¹J_(C-P)=4.07 Hz, C5), 13.08 (C1) ppm;³¹P NMR (121.45 MHz, CDCl₃, δ): 26.92 ppm; HRMS-DART (m/z): [M⁺]+calculated for C₂₃H₅₁NO₃P, 420.3601. found, 420.3608.

α-Amino Bisphosphonic Acid Antimicrobials (α-ABPQ).

Synthesis of α-Amino Bisphosphonic Quats-Via Double Kabachnik FieldsReaction Example 3 Tetraethyl(((3-bromopropyl)azanediyl)bis(methylene))bis(phosphonate) (4)

To a 20 mL glass screw cap vial, equipped with a magnetic stir bar wasadded diethylphosphite (2.77 mL, 21.56 mmol, 2.2 eq.) and the vial wasplaced on ice meanwhile 3-aminopropyl-1-bromide hydrobromide (˜2.5 g,˜11 mmol) was treated with KOH (6N, 6 g in 20 mL) and stirred at 0° C.until a yellow oil appeared (˜5 min). The mixture was then extractedwithout solvent, collecting the upper yellow layer of the free baseaminopropyl-1-bromide (incompletely dry by NMR, 50% water present). Theamine (1.350 g, 9.78 mmol) was added to the vial containing diethylphosphite and cooled at 0-5° C. (ice bath). To the chilled, stirredsolution was added formalin, dropwise (37%, 2.12 mL, 25.43 mmol, 2.6eq.) over 10 min while maintaining the reaction temp under 10° C., thenwarming the mixture to room temperature for 30 min, and finally to 100°C. for 1 h. The reaction was diluted with 0.2N NaOH (˜300 mg in 40 mL)and extracted with CHCl₃ (1×30 mL, 1×10 mL), the organic layer wasseparated, washed with brine (1×20 mL) and dried over anhydrous MgSO₄filtered and concentrated to afford a yellow oil. The title compound wasin poor yield, however analysis by ¹H NMR revealed >98% purity andrequired no further purification. Yield 20.9% (0.7658 g); TLC (5% MeOHin EtOAc), R_(f)=0.48; ¹H NMR (400 MHz, CDCl₃, δ): 4.17-4.07 (m, 8H,H6), 3.47 (t, 2H, J=6.7 Hz, H5), 3.14 (d, 4H, J=8.5 Hz, H4), 2.93 (t,2H, J=6.6 Hz, H3), 2.00 (q, 2H, J=6.58 Hz, H2), 1.31 (t, 12H, J=7.1 Hz,H1); ¹³C NMR (100 MHz, CDCl₃, δ): 61.9 (t, ²J_(C-P)=3.36 Hz, C6), 55.08(C3), 49.43 (C4), 31.09 (C5), 30.96 (C2), 16.49 (t, ³J_(C-P)=2.94 Hz,C1); ³¹P NMR (121.45 MHz, CDCl₃, δ): 24.60 ppm.

Example 4 Tetraethyl(((3-chloropropyl)azanediyl)bis(methylene))bis(phosphonate) (5)

To a 20 mL glass screw cap vial, equipped with a magnetic stir bar wasadded diethylphosphite (2.86 g, 20.74 mmol, 2.0 eq.). The vial wasplaced on ice to cool. In a separate beaker, 3-aminopropyl-1-chloridehydrochloride (2.0 g, 11.4 mmol) was treated with NaOH (˜12 N, 2 g in 5mL) and stirred at 0° C. until a yellow oil appeared (˜5 min). Themixture was then extracted without solvent, adding the upper yellowlayer of the free base 3-aminopropyl-1-chloride to the vial containingdiethyl phosphite cooled to 0-5° C. (ice bath). To the chilled solutionwas added formalin, dropwise, via syringe (37%, 2.15 mL, 25.79 mmol, 2.5eq.) over 10 min maintaining the reaction temp under 10° C. The mixturewas then warmed, with stirring, to room temperature for 10 min, thenheated to 100° C. for 30 min. Excess formaldehyde and water via rotovapand the crude material purified by Dry Column Vacuum Chromatography(DCVC) on silica gel (20 g silica, 3.5 cm×4.5 cm) eluting with 80 mLEtOAc and collecting 50 mL (20% MeOH/EtOAc). Yield 50% (2.03 g); TLC(10% MeOH in EtOAc), R_(f)=0.70; ¹H NMR (400 MHz, CDCl₃, δ): 4.18-4.09(m, 8H, H6), 3.62 (t, 2H, J=6.6 Hz, H5), 3.17 (d, 4H, J=8.6 Hz, H4),2.97 (t, 2H, J=6.6 Hz, H3), 2.00 (p, 2H, J=6.64 Hz, H2), 1.33 (t, 12H,J=7.1 Hz, H1); ¹³C NMR (100 MHz, CDCl₃, δ): 61.84 (p, ²J_(C-P)=3.58 Hz,C6), 53.93 (t, ³J_(C-P)=7.44 Hz, C5), 50.18 (dd, =6.08 Hz,¹J_(1C-P)=6.00 Hz, C4), 42.47 (C3), 30.77 (C2), 16.45 (t, ³J_(C-P)=2.94Hz, C1); ³¹P NMR (121.45 MHz, CDCl₃, δ): 24.40 ppm.

Example 5 Tetraethyl(((3-hydroxypropyl)azanediyl)bis(methylene))bis(phosphonate) (6)

This compound has been previously reported in: Cavero, E., Zablocka, M.,Caminade, A. & Majoral, J. P. Design of Bisphosphonate-TerminatedDendrimers. Eur. J. Org. Chem., 2759-2767 (2010); Chougrani, K.,Boutevin, B., David, G., Seabrook, S. & Loubat, C. Acrylate basedanticorrosion films using novel bis-phosphonic methacrylates. J. Polym.Sci., Part A: Polym. Chem. 46, 7972-7984 (2008); and Chougrani, K.,Boutevin, B., David, G. & Boutevin, G. NewN,N-amino-diphosphonate-containing methacrylic derivatives, theirsyntheses and radical copolymerizations with MMA. Eur. Polym. J. 44,1771-1781 (2008). To a 20 mL glass screw cap vial, equipped with amagnetic stir bar was added diethylphosphite (2.86 g, 20.74 mmol, 2.0eq.) and 3-amino-1-propanol (0.768 g, 10.24 mmol,) and the mixturecooled to 0-5° C. (ice bath). To the chilled solution was addedformalin, dropwise, via syringe (37%, 2.15 mL, 25.79 mmol, 2.5 eq.) over10 min maintaining the reaction temp under 10° C. The mixture waswarmed, with stirring, to room temperature for 30 min, then heated to100° C. for 60 min. Excess formaldehyde and water were removed viarotovap and the crude material purified by Dry Column VacuumChromatography (DCVC) on silica gel (20 g silica, 3.5 cm×4.5 cm) elutingwith 100 mL EtOAc (20% MeOH/EtOAc). Yield 50% (2.03 g); TLC (10% MeOH inEtOAc), R_(f)=0.50; ¹H NMR (400 MHz, CDCl₃, δ): 4.18-4.09 (m, 8H, H6),3.62 (t, 2H, J=6.6 Hz, H5), 3.17 (d, 4H, J=8.6 Hz, H4), 2.97 (t, 2H,J=6.6 Hz, H3), 1.61 (p, 2H, J=5.56 Hz, H2), 1.32 (t, 12H, J=7.1 Hz, H1)ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ): 25.0 ppm.

Example 6 3-(bis((diethoxyphosphoryl)methyl)amino)propyl4-methylbenzenesulfonate (7)

To a flame dried and evacuated 25 mL round bottom flask, equipped with amagnetic stir bar was added sequentially trimethylamine hydrochloride(0.045 g, 0.24 mmol, 0.24 eq.), DCM (1 mL), triethylamine (0.58 mL, 2.5mmol, 2.5 eq) the alcohol (0.375 g, 1 mmol) and the solution cooled to0° C. in an ice bath. To the chilled, stirred solution was added,dropwise, tosyl chloride, anhydrous DCM (2 mL) and the cloudy yellowmixture was stirred for 1 hr at room temperature at which point TLCshowed disappearance of the starting amine (5% MeOH in EtOAc, 10 mL).The reaction was diluted with water (1×15 mL) and extracted with DCM (10mL total), the aqueous layer was re-extracted with EtOAC (15 mL) and thecombined organic layers were dried over MgSO₄, filtered and evaporatedto give a yellow oil. The crude material was purified by flashchromatography on silica gel (20 g silica, 1.5 cm i.d) with gradientelution: 100% EtOAc (35 mL) then 5% MeOH:EtOAc (90 mL) to obtain thetitle compound as a yellow oil. Yield 56.7% (0.3003 g); TLC (5% MeOH inEtOAc), R_(f)=0.42; ¹H NMR (400 MHz, CDCl₃, δ): 7.76 (d, 2H, J=8.24 Hz,H9), 7.32 (d, 2H, J=8.04 Hz, H8), 4.13-4.05 (m, 10H, H7+H6), 3.08 (d,4H, J=8.40 Hz, H5), 2.83 (t, 2H, J=6.70 Hz, H4), 2.42 (s, 3H, H3), 1.81(t, 2H, J=6.65 Hz, H2), 1.30 (t, 12H, J=7.08 Hz, H1); ¹³C NMR (100 MHz,CDCl₃, δ): 44.67 (C11), 133.19 (C10), 129.81 (C8), 127.83 (C9), 68.50(C7), 61.86 (t, ²J_(C-P)=3.19 Hz, C6), 52.67 (dd, ¹J_(C-P)=6.02 Hz, C5),52.67 (C4), 27.22 (C2), 21.57 (C3), 16.46 (t, ³J_(C-P)=2.78 Hz, C1) ppm;³¹P NMR (121.45 MHz, CDCl₃, δ): 24.56 ppm.

Example 7 Tetraethyl(((3-(dimethylamino)propyl)azanediyl)bis(methylene)) bis(phosphonate)(8)

To a 20 mL glass screw cap vial equipped with a magnetic stir barcontaining the bromo amino bisphosphonate (0.954 g, 1.8 mmol) was addedNHMe₂ (5.6 M in EtOH, 2.5 mL, excess) followed by H₂O (0.5 mL) and theclear mixture was stirred at reflux sealed for 1.5 hr, at which pointTLC showed disappearance of the starting material (1% NH₄ ⁺OH⁻ inAcetone, 10 mL, R_(f)=0.95). The cooled yellow reaction diluted withwater (1×20 mL, pH was 11) and extracted with CHCl₃ (2×30 mL), driedover MgSO₄, filtered and evaporated to give an orange oil. The titlecompound was isolated >98% purity (¹H and ³¹P NMR) and required nofurther purification. Yield 62% (0.4461 g); TLC (1% NH₄ ⁺OH⁻ in Acetone,10 mL) or (20% MeOH (6% NaBr):MeCN, R_(f)=0.47; ¹H NMR (400 MHz, CDCl₃,δ): 4.15-4.06 (m, 8H, H7), 3.11 (d, 4H, J=8.9 Hz, H6), 2.80 (t, 2H,J=6.8 Hz, H5), 2.27 (t, 2H, J=7.5 Hz, H4), 2.18 (s, 6H, H3), 1.61 (p,2H, J=7.15 Hz, H2), 1.28 (t, 12H, J=7.0 Hz, Hp ppm; ¹³C NMR (100 MHz,CDCl₃, δ): 61.8 (t, ²J_(CP)=3.3 Hz, C7), 57.24 (C4), 55.03 (C5), 50.92(dd, ¹J_(CP)=7.1 Hz, C6), 49.36 (dd, ¹J_(CP)=6.7 Hz, C6), 45.48 (C3),25.65 (C2), 16.48 (t, ³J_(CP)=2.8 Hz, C1) ppm; ³¹P NMR (121.45 MHz,CDCl₃, δ): 24.89 ppm.

Example 8N-(3-(bis((diethoxyphosphoryl)methyl)amino)propyl)-N,N-dimethyloctadecan-1ammoniumBromide (9)

To a flame dried and evacuated 20 mL screw cap vial, equipped with amagnetic stir bar was added a mixture of bromoaminobisphosphonate (0.2g, 0.51 mmol) and DMOA (0.143 g, 0.6 mmol, 1.19 eq.) was which wassealed and heated to 100° C. on a sand batch. After 1 hr, TLC showed thedisappearance of the starting amine (5% MeOH in EtOAc, 10 mL). Themixture was partitioned between hexanes (˜7 mL) and MeOH/H₂O (4:1, 5mL), the bottom yellow methanolic layer was separated and concentrated(2×5 mL ACN to azeotrope excess water) to afford a yellow oily solid(0.3082 g). The crude material was purified by Dry Column VacuumChromatography (DCVC) on silica gel (20 g silica, 3.5 cm×4.5 cm)pre-washed with 60 mL 20% MeOH (NaBr 6%):ACN then eluting with the sameeluent (1st 40 mL removed upper R_(f) impurity, product was obtained inthe next 7 fractions total 95 mL) as a yellow oil after filtering offNaBr through a pad of Celite washing with CHCl₃. Yield 46.1% (0.162 g).TLC (20% MeOH (NaBr 6%):ACN), R_(f)=0.5; ¹H NMR (400 MHz, CDCl₃, δ)4.15-4.08 (m, 8H, H11), 3.72-3.69 (m, 2H, H10), 3.55-3.51 (m, 2H, H9),3.33 (s, 6H, H8), 3.12-3.08 (m, 4H, H7), 2.99-2.97 (m, 2H, H6), 2.0-1.98(m, 2H, H5), 1.74-1.71 (m, 2H, H4), 1.24-1.20 (br m, 42H, H2, H3,overlap) 0.88-0.83 (m, 3H, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ):62.15-62.0 (overlap, C7, C9, C10, C11), 51.1 (C6, C8 overlap), 31.91 (C2overlap), 29.67-29.27 (C2 overlap), 22.84 (C4), 22.67 (C5), 16.58-16.50(m, J_(CP)=unresolved, C3), 14.10 (C1) ppm; ³¹P NMR (121.45 MHz, CDCl₃,δ) 24.40 ppm. HRMS-DART (m/z): [M⁺] calculated for C₃₃H₇₃N₂O₆P₂,655.4937. found, 655.4938.

Synthesis of α-Amino Bisphosphonic Quats-Via Triazinane Intermediate.

Example 9 3,3′,3″-(1,3,5-triazinane-1,3,5-triyl)tris(propan-1-ol) (10)

To a 125 mL round bottom flaks, formalin (0.813 mL, 10 mmol) was addedto a solution of 3-amino-1-propanol (0.751 g, 10 mmol) in MeCN (10 mL).The reaction was stirred at room temperature overnight. Evaporation ofvolatiles followed by (DCVC) on silica gel (20 g silica, 3.5 cm×4.5 cm)eluting with 5% NH₄ ⁺OH⁻ in acetone (50 mL) then collecting (150 mL)provided pure product. Yield 92% (0.8 g). TLC (5% NH₄ ⁺OH⁻ in acetone,10 mL), R_(f)=0.3; ¹H NMR (400 MHz, CDCl₃, δ): 4.37 (s, 6H, H5), 3.84(t, 6H, J=7.0 Hz, H4), 3.71 (s, 3H, H3), 2.97 (t, 6H, J=5.6 Hz, H2),1.60 (q, 6H, J=5.40 Hz, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 83.06(C4), 68.12 (C3), 47.78 (C2), 22.45 (C1) ppm.

Example 103,3′,3″-(1,3,5-triazinane-1,3,5-triyl)tris(N,N-dimethylpropan-1-amine)(11)

To a 125 mL round bottom flask, paraformaldehyde (1.652 g, 55 mmol, 1.1eq.) was added to a solution of N,N-dimethylpropane-1,3-diamine (6.29mL, 50 mmol) in toluene (15 mL). The reaction was refluxed using adean-stark trap for 1.5 hr. Toluene was evaporated and a portion of theresidue (1.9757 g) was partitioned between CHCl₃ (15 mL) and water (5mL). The organic layer was separated, dried with MgSO₄ and concentratedto give a clear oil. Yield 66% (1.3067 g). TLC (20% MeOH in EtOAc, 10mL), R_(f)=0.05; ¹H NMR (400 MHz, CDCl₃, δ): 3.29 (brs, 6H, H5), 2.40(t, 6H, J=7.5 Hz, H4), 2.25 (t, 6H, J=7.5 Hz, H3), 2.18 (s, 18H, H2),1.59 (p, 6H, J=7.5 Hz, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 74.65 (C5),57.83 (C3), 50.78 (C4), 45.54 (C2), 25.88 (C1) ppm.

β-Amino Bisphosphonic Acid Antimicrobials (β-ABPQ) Example 11 Tetraethyl(((3-hydroxypropyl)azanediyl)bis(ethane-2,1-diyl))bis(phosphonate) (12)

This compound has been previously reported in: Pothayee, N. et al.Synthesis of ‘ready-to-adsorb’ polymeric nanoshells for magnetic ironoxide nanoparticles via atom transfer radical polymerization. Polymer52, 1356-1366 (2011). To a 25 mL round bottom flask equipped with amagnetic stir bar, was added a stirred solution of the primary amine(0.448 g, 5.9 mmol) in distilled water (5 ml) at room temperature. Twoequivalents of diethyl vinylphosphonate (1.637 g, 12.03 mmol, 2.01 eq.)was then added and the reaction stirred at room temperature overnight.The reaction was transferred to a 125 mL round bottom flask along with30 mL MeCN and evaporated to a clear oil (2.1446 g, containing ≈7%starting material by ³¹P NMR). The crude material was purified by DryColumn Vacuum Chromatography (DCVC) on silica gel (20 g silica, 3.5cm×4.5 cm) eluting with 30% MeOH:EtOAc (240 mL). The fractionscontaining the title compound were filtered through a pad of Celiteevaporated to obtain the title compound as a clear oil. Yield 95%(1.9687 g); TLC (30% MeOH:EtOAc), R_(f)=0.33; ¹H NMR (400 MHz, CDCl₃,δ): 4.16-4.03 (m, 8H, H7), 3.75-3.67 (m, 3H, H6), 2.83-2.75 (m, 4H, H5),2.64-2.59 (m, 2H, H4), 1.97-1.86 (m, 4H, H3), 1.68 (q, 2H, J=5.58 Hz,H2), 1.31 (t, 12H, J=7.06 Hz, HD ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ):30.00 ppm.

Example 12 Tetraethyl(((3-(dimethylamino)propyl)azanediyl)bis(ethane-2,1-diyl)) Bbis(phosphonate) (13)

Synthesized from alcohol via mesylate and dimethylamine, see Tetraethyl(((3-(dimethylamino)propyl)azanediyl)bis(methylene))bis(phosphonate)procedure; ¹H NMR (400 MHz, CDCl₃, δ): 4.11-3.99 (m, 8H, H8), 2.76-2.69(m, 4H, H7), 2.40 (t, 2H, J=7.12 Hz, H6), 2.22 (t, H5, J=7.14 Hz, H5),2.16 (s, 6H, H4), 1.91-1.81 (m, 4H, H3), 1.60-1.53 (m, H2, 2H), 1.28 (t,H1, J=7.04 Hz) ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ): 30.57 ppm.

Bisphosphonic Acid Antimicrobials (BPQ).

Syntheses of Bisphosphonic Quats—Direct Alkylation ofTetraethylmethylene Bisphosphonate.

Syntheses of Bisphosphonic Quats—Via α-Mesylate Example 13 Diethyl(4-(1,3-dioxoisoindolin-2-yl)-1-hydroxybutyl) Phosphonate (14)

A 25 mL round bottom flask, equipped with a magnetic stir bar and acondenser was charged with the aldehyde (2.281 g, 10.5 mmol),diethylphosphonate (1.523 g, 11.03 mmol, 1.05 eq.), K₂CO₃ (0.073 g, 0.53mmol, 0.05 eq.) and MeCN (5 mL). The heterogeneous solution was stirredat 60° C. for 15 min at which point TLC showed disappearance of thestarting aldehyde (60% EtOAc in hexanes, 10 mL). The reaction was cooledto 0° C., filtered and evaporated. The resulting yellow oil solidifiedunder high vacuum (10 min) and was recrystallized from hot EtOAc (5 mL)after cooling for 20 min at 0° C. Yield 69.1% (2.5787 g); TLC (60% EtOAcin hexanes), R_(f)=0.2; ¹H NMR (400 MHz, CDCl₃, δ): 7.83-7.79 (m, 4H,H8), 7.71-7.64 (m, 4H, H7), 4.18-4.07 (m, 4H, H6), 3.89 (quintet, J=4.59Hz, 1H, H5), 3.77-3.66 (m, 2H, H4), 2.05-1.95 (m, 2H, H3), 1.87-1.68 (m,2H, H2), 1.29 (t, J=7.08 Hz, 6H, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ):168.37 (C10), 133.90 (C9), 132.10 (C7), 123.18 (C8), 68.12 (C5), 62.65(q²J_(C-P)=7.3 Hz, C6), 37.52 (C2), 28.43 (d, ¹J_(C-P)=1.45 Hz, C5),25.02 (C3), 24.96 (C4), 16.46 (d, ³J_(C-P)=5.20 Hz, C1) ppm; ³¹P NMR(121.45 MHz, CDCl₃, δ): 24.64 ppm.

Example 14 1-(diethoxyphosphoryl)-4-(1,3-dioxoisoindolin-2-yl) ButylMethanesulfonate (15)

To a flame dried and evacuated 50 mL round bottom flask, equipped with amagnetic stir bar was added sequentially trimethylamine hydrochloride(0.062 g, 0.62 mmol, 0.20 eq.), DCM (2 mL), triethylamine (0.65 mL, 4.63mmol, 1.5 eq.) and the alcohol (1.097 g, 3.09 mmol) and the solution wascooled to 0° C. in an ice bath. To the chilled stirred solution wasadded, dropwise, mesyl chloride (0.25 mL, 3.70 mmol, 1.2 eq.) inanhydrous DCM (2 mL) and the cloudy yellow mixture was stirred for 20min at room temperature at which point TLC showed disappearance of thestarting amine (10% MeOH in EtOAc, 10 mL). The reaction was diluted withwater (1×10 mL) and extracted with DCM (2×5 mL total), the combinedorganic layers were dried over MgSO₄, filtered and evaporated to give ayellow oil. The crude product (1.409 g) containing traces of DCM andexcess mesyl chloride by ¹H NMR, was placed under high vacuum at 60° C.for 1 hr. Yield 93% (1.2013 g); TLC (10% MeOH in EtOAc), R_(f)=0.5; ¹HNMR (400 MHz, CDCl₃, δ): 7.85-7.81 (m, 2H, H9), 7.73-7.70 (m, 2H, H8),4.94-4.88 (m, 1H, H7), 4.20-4.15 (m, 4H, H6), 3.77-3.70 (m, 2H, H5),3.15 (s, 3H, H4), 1.95-1.82 (m, 4H, H2, H3), 1.41-1.25 (m, 6H, H1) ppm;¹³C NMR (100 MHz, CDCl₃, δ): 168.26 (C11), 133.98 (C10), 132.08 (C9),123.22 (C8), 74.79 (C7), 63.31 (q, ²J_(C-P)=7.3 Hz, C6), 52.56 (C2),39.11 (C4), 27.58 (C3), 24.45 (d, ²J_(C-P)=11.67 Hz C5), 16.45(²J_(C-P)=5.20 Hz, C1) ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ): 17.63 ppm.

Syntheses of Bisphosphonic Quats—Via Michael Addition to aVinylbisphosphonate.

Example 15 Tetraethyl ethene-1,1-diylbis(phosphonate) (16)

This compound has been previously reported in: Gebbia, N., Simoni, D.,Dieli, F., Tolomeo, M. & Invidiata, F. P. Geminal bisphosphonates, theirpreparation and their use in the field of oncology. PCT Int. Appl., 38(2009); and Simoni, D. et al. Design, Synthesis, and BiologicalEvaluation of Novel Aminobisphosphonates Possessing an in Vivo AntitumorActivity Through a T Lymphocytes-Mediated Activation Mechanism. J. Med.Chem. 51, 6800-6807 (2008). A 50 mL round bottom flask was charged withparaformaldehyde (6.3 g, 200 mmol, 4.0 eq.) and diethylamine (5.2 mL, 50mmol, 1 eq.) in methanol (125 mL) and the mixture was stirred underreflux until a clear solution was obtained (˜5 min). Tetraethylmethylenebisphosphonate was added via syringe (12.4 mL, 50 mmol, 1.0 eq.) and thesolution was refluxed overnight (24 hr). The clear solution wasconcentrated in vacuo and then re-evaporated from toluene (2×10 mL)completely removing residual MeOH to give the intermediate methyl etheras a clear oil. The residue was dissolved in toluene (100 mL), treatedwith p-toluenesulphonic acid (38 mg, 0.02 mmol), and refluxed through aDean-Stark trap overnight. The orange solution was concentrated invacuo, dissolved in chloroform (50 mL), washed with water (2×10 mL),dried over MgSO₄, and concentrated in vacuo. A portion of the orange oil(6 g) was further distilled under high vacuum. Yield 90% (5.4 g); TLC(EtOAc), R_(f)=0.2; ¹H NMR (400 MHz, CDCl₃, δ): 7.02-6.86 (m, H3, 2H),4.10-4.05 (m, H2, 8H), 1.34-1.21 (m, H1, 12H) ppm; ¹³C NMR (100 MHz,CDCl₃, δ): 149.04 (m, C4), 133.77-129.71 (m, C3), 62.54 (t, ²J_(CP)=2.88Hz, C2), 16.17 (t, ³J_(CP)=3.15 Hz, C1) ppm; ³¹P NMR (121.45 MHz, CDCl₃,δ): 21.0 ppm.

Syntheses of Bisphosphonic Quats—Via Phosphorylation of aMono-Phosphonate.

Example 16 Diethyl (4-(1,3-dioxoisoindolin-2-yl)butyl) Phosphonate (17)

This compound has been previously reported in Hara, T., Durell, S. R.,Myers, M. C. & Appella, D. H. Probing the Structural Requirements ofPeptoids That Inhibit HDM2-p53 Interactions. J. Am. Chem. Soc. 128,1995-2004 (2006). To a flame dried 50 mL round bottom flask equippedwith a reflux condenser was added N-(4-Bromobutyl)-phthalimide (5 g,17.7 mmol, 1.0 eq.) followed by triethylphosphite (18.24 mL, 106.3 mmol,6 eq.) and the mixture was refluxed overnight (175° C.) using a sandbath. The reaction was then cooled to room temperature and excesstriethylphosphite was vacuum distilled using a shortpath distillationhead attached to a Schlenk line. Once all of the excesstriethylphosphite stopped distilling, the title compound was placedunder high vacuum (˜30 min) until it solidified. Furtherrecrystallization from EtOAc (5 mL) at −20° C. provided pure product.Colourless crystals. Yield: 90% (5.4263 g). TLC (5% MeOH: EtOAc),R_(f)=0.90; ¹H NMR (400 MHz, CDCl₃, δ): 7.82-7.77 (n, 2H, H8), 7.70-7.66(m, 2H, H7), 4.11-3.98 (m, 4H, H6), 3.66 (m, J=7.0 Hz, 2H, H5),1.81-1.71 (m, 4H, H4, H3), 1.67-1.56 (m, 2H, H2), 1.27 (t, J=7.1 Hz, H1)ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 168.29 (C10), 133.91 (C9), 132.06(C7), 123.18 (C8), 61.48 (d, ²J_(C-P)=6.5 Hz, C6), 37.23 (d,¹J_(C-P)=1.33 Hz, C5), 29.25 (d, ²J_(C-P)=16.77 Hz C4), 24.44 (C2),19.81 (d, ³J_(C-P)=5.01 Hz, C3), 16.42 (d, ³J_(C-P)=6.01 Hz, C1) ppm;³¹P NMR (121.45 MHz, CDCl₃, δ): 31.48 ppm.

Example 17 2-(3-bromopropoxy)tetrahydro-2H-pyran (18)

This compound has been previously reported in: Pinchuk, A. N. et al.Synthesis and Structure-Activity Relationship Effects on the TumorAvidity of Radioiodinated Phospholipid Ether Analogues. J. Med. Chem.49, 2155-2165 (2006). To a stirred solution inside a 125 mL round bottomflask containing 3-bromo-1-propanol (6.95 g, 50 mmol, 1 eq.) in DCM (25mL) was added 3,4-dihydropyran (5.93 mL, 65 mmol, 1.3 eq.). The mixturewas stirred overnight at room temperature at which point TLC showeddisappearance of 3-bromo-1-propanol (20% EtOAc in hexanes, 10 mL,KMnO₄). The reaction was evaporated and the crude material was purifiedby flash chromatography on silica gel (20 g silica, 1.5 cm i.d) elutingwith 10% EtOAc:hexanes (100 mL) to obtain the title compound as a clearoil. Yield 86.4% (9.637 g); TLC (20% EtOAc in hexanes), R_(f)=0.85; ¹HNMR (400 MHz, CDCl₃, δ): 4.59 (t, 1H, J=3.52 Hz, H7), 3.90-3.81 (m, 2H,H6), 3.55-3.47 (m, 4H, H4+H5), 2.16-2.08 (m, 2H, H3), 1.90-1.64 (m, 2H,H2), 1.57-1.50 (m, 4H, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 98.90 (C7),64.88 (C6), 62.26 (C5), 32.90 (C3), 30.59 (d, ²J=6.04 Hz, C4), 25.41(C2), 19.48 (C1) ppm.

Example 18 Diethyl (3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)phosphonate(19)

This compound has been previously reported in: Voigt, M. et al. SurfaceFunctionalization of ZnO Nanorods with C60 Derivatives CarryingPhosphonic Acid Functionalities. J. Phys. Chem. C 115, 5561-5565 (2011).To a 20 mL conical round bottom flask was added the THP protectedbromopropylalcohol (4.55 g, ˜20 mmol) followed by excess triethylphosphite (10.0 mL, 60.0 mmol, 3.0 eq.). The reaction was heated atreflux (175° C.) overnight. Excess triethyl phosphite was vacuumdistilled at reduced pressure providing the pure product as a clear,viscous oil. Yield 89% (5 g). ¹H NMR (400 MHz, CDCl₃, δ): 4.57 (t, 2H,J=3.54 Hz, H8), 4.17-4.04 (m, 4H, H7), 3.86-3.72 (m, 2H, H6), 3.52-3.40(m, 2H, H5), 1.93-1.77 (m, 4H, H4+H3), 1.73-1.67 (m, 4H, H2), 1.31 (t,6H, J=7.04 Hz, H1) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 98.86 (C8), 64.85(C6), 62.21 (C5), 32.90 (C3), 30.66 (C4), 25.43 (C2), 19.71 (C1) ppm.

Example 19 Diethyl (4-(dimethylamino)butyl)phosphonate (20)

A mixture of diethyl (4-bromobutyl)phosphonate (5.0 g, 18.3 mmol) withNHMe₂ (5.6 M in EtOH, 10 mL, excess) was placed, with a magneticstirring bar, into a 20 ml glass reaction tube and sealed. The reactionmixture was heated in the Biotage® Initiator Microwave Synthesizer at110° C. (5 min). Volatiles were removed on a rotovap and the crudematerial was purified by Dry Column Vacuum Chromatography (DCVC) onsilica gel (50 g silica, 3:5 cm×5.5 cm) eluting first with 150 mL (10%MeOH/acetone) collecting 250 mL (10% MeOH/10% NH₄ ⁺OH⁻/80% acetone).Yield 81% (3.55 g); TLC (20% NH₄ ⁺OH⁻/acetone), Rf=0.50; ¹H NMR (400MHz, CDCl₃, δ): 4.06-3.92 (m, 4H, H7), 2.85 (t, 2H, J=7.96 Hz, H6), 2.62(s, 6H, H5), 1.83-1.53 (m, 6H, H4-H2 overlap), 1.22 (t, 6H, J=7.04 Hz,Hp ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 61.71 (d, ²J_(CP)=6.60 Hz, C7),57.68 (C6), 43.58 (C5), 25.68 (t, ¹J_(CP)=14.07 Hz, C2), 24.13 (C4),19.90 (d, ²J_(CP)=4.60 Hz, C3), 16.41 (d, ³J_(CP)=6.22 Hz, C1) ppm; ³¹PNMR (121.45 MHz, CDCl₃, δ): 30.89 ppm.

Syntheses of Bisphosphonic Quats—Via Triethylorthoformate

Example 20 Tetraethyl dimethylaminomethylenediphosphonate (21)

This compound has been previously reported in: O'Boyle, N. M. et al.Synthesis, evaluation and structural studies of antiproliferativetubulin-targeting azetidin-2-ones. Bioorg. Med. Chem. 19, 2306-2325(2011). To a chilled solution of dimethylformamide (3.87 mL, 50 mmol) inDCM (75 mL) was added dropwise with stirring a solution of oxalylchloride (25 mL, 2M in DCM, 50 mmol). Following addition, the mixturewas allowed to warm to room temperature and stirred for 1 h. Triethylphosphite (18.77 mL, 109.5 mmol, 2.19 eq.) was then added dropwise withstirring. After 1 hr the mixture was concentrated under reducedpressure. The product was obtained as a yellow oil in 75.5% yield. ¹HNMR (400 MHz, CDCl₃, δ): 4.21-4.14 (m, 8H, H4), 3.22 (dt, 1H, ¹J=24.98Hz, ²J=24.98 Hz, H3), 2.58 (s, 6H, H2), 3.18 (dt, 12H, J=7.07 Hz, J=7.06Hz, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃, δ): 62.70 (t, ¹J_(C-P)=3.05 Hz,C3), 62.40 (t, ²J_(C-P)=3.61 Hz, C4), 44.11 (t, ³J_(C-P)=4.71 Hz, C2),16.39 (q, ³J_(C-P)=3.01 Hz, C1) ppm; ³¹P NMR (121.45 MHz, CDCl₃, δ):19.15 ppm.

Multidentate Phosphonic Acid Antimicrobial Structures Bisphosphonic AcidAntimicrobials N-(4,4-diphosphonobutyl)-N,N-dimethyloctadecan-1ammoniumBromide. (22)

N-(3-(bis(phosphonomethyl)amino)propyl)-N,N-dimethyloctadecan-1ammoniumBromide (23)

N-(3-(bis(2-phosphonoethyl)amino)propyl)-N,N-dimethyloctadecan-1ammoniumBromide (24)

Trisphosphonic Acid AntimicrobialsN,N-dimethyl-N-(4,4,4-triphosphonobutyl)octadecan-1ammonium Bromide (25)

Tris Ether Phosphonic Acid-1-N,N-dimethyloctadecan-1ammonium Bromide.(26)

N-(3-((1,3-bis(3-phosphonopropoxy)-2-((3-phosphonopropoxy)methyl)propan-2-yl)amino)-3-oxopropyl)-N,N-dimethyloctadecan-1ammonium Bromide. (27)

Tetraphosphonic Acid AntimicrobialsN-(3-(bis(2,2-diphosphonoethyl)amino)propyl)-N,N-dimethyloctadecan-1ammoniumBromide. (28)

N-(3-(bis(2-(bis(2-phosphonoethyl)amino)ethyl)amino)propyl)-N,N-dimethyloctadecan-1ammonium Bromide. (29)

Dansyl-Phosphonic Acid Antimicrobials—UV Detection.

Synthesis of Dansylphosphonic Acid Quats (DPQ) Example 215-(dimethylamino)-N-(3-(dimethylamino)propyl)naphthalene-1-sulfonamide(30)

This compound has been previously reported in: Wang, X. & Schneider, H.Binding of dansylamide derivatives to nucleotides and nucleic acids. J.Chem. Soc., Perkin Trans. 2, 1323-1328 (1998). To a flame dried 500 mLround bottom flask with a reflux condenser connected to an inertatmosphere manifold anhydrous DCM (300 mL) was added followed by dansylchloride (10.0 g, 37.07 mmol), triethylamine (˜8 mL, 55.61 mmol). Whilethe solution was stirring at room temperature,3-(dimethylamino)propylamine (7.0 ml, 55.61 mmol) was added drop wisevia an inert syringe resulting in a colour change from orange tolime-green. After stirring for 1 h —HCl (g) was bubbled through thesolution until pH 2 was reached. The resulting mixture was evaporated todryness, then re-dissolved in saturated brine water (100 mL) andbasified to pH 11 with 6N NaOH (15 mL) at 0° C. until white-yellowprecipitate was observed. The mixture was refrigerated overnightenhancing further precipitation of product. The precipitate was filteredwashing with water and the filtrate was extracted with DCM (500 mL) andevaporated to dryness to afford a white solid in 97% yield (12.1 g).(Recrystallized using 80% EtOH/H₂O). Mp=122-124° C.; TLC (5% NH₄⁺OH⁻:Acetone), R_(f)=0.72: ¹H NMR (400 MHz, CDCl₃, δ): 8.52 (ddd, ¹J=1.5Hz, ²J=1.5 Hz, ³J=8.5 Hz, 1H, H9), 8.31 (ddd, ¹J=1.0 Hz, ²J=1.0 Hz,³J=8.5 Hz, 1H, H6), 8.23 (dd, ¹J=1.5 Hz, ²J=7.0 Hz, 1H, H11), 7.50-7.58(m, 2H, H(5, 10)), 7.18 (dd, ¹J=1.0, ²J=7.5, 1H, H4), 2.97 (t, J=5.5 Hz,2H, H14), 2.90 (s, 6H, H(1,2)), 2.22 (t, J=5.5 Hz, 2H, H16), 2.14 (s,6H, H(17,18)), 1.57 (p, ¹J=5.8 Hz, 2H, H15) ppm; ¹³C NMR (100 MHz,CDCl₃, δ): 151.9 (C3), 134.7 (C12), 129.98-129.65 (m, overlap, C5, C7,C9, C10, C11), 123.1 (C6), 119.0 (C8), 115.0 (C4), 59.6 (C16), 45.4 (C1,C2, C17, C18), 44.5 (C14), 24.6 (C15) ppm. HRMS-DART (m/z): [M⁺]calculated for C₁₇H₂₆N₃O₂S₁, 336.1736. found, 336.1745.

Example 223-(diethoxyphosphoryl)-N-(3-(5-(dimethylamino)naphthalene-1-sulfonamido)propyl)-N,N-dimethylpropan-1-ammoniumBromide (31)

To a flame dried 20 mL glass vial, ACN (3 mL) was added followed by5-(dimethylamino)-N-(3-(dimethylamino)propyl)-naphthalene-1-sulfonamide(335.46 mg, 1 mmol). While stifling diethyl(3-bromopropyl)phosphonate(˜0.4 mL, 2 mmol) was added via an inert syringe, and the vial wascapped. The solution was stirred for 48 hr at 110° C., after which thesolution turned to pale-yellowish oil. The solution was cooled to roomtemperature, washed with Et₂O (3×10 mL) to remove soluble impuritiesfrom the crude product. The product was further dried using rotaryevaporator resulting in orange gummy oil in 70% yield (416.5 mg)Mp=34-36° C.; ¹H NMR (400 MHz, CDCl₃, δ): 8.52 (ddd, ¹J=1.5 Hz, ²J=1.5Hz, ³J=8.5 Hz, 1H, H9), 8.31 (ddd, ¹J=1.0 Hz, 2J=1.0 Hz, ³J=8.5 Hz, 1H,H6), 8.23 (dd, =1.5 Hz, ²J=7.0 Hz, 1H, H11), 7.50-7.58 (m, 2H, H(5,10)), 7.18 (dd, ¹J=1.0, ²J=7.5, 1H, H4), 4.12-4.03 (m, 4H, H(22,23)),3.68-3.57 (m, 4H, H(16,19)), 3.18 (s, 6H, H(17, 18)), 3.10-3.03 (m, 2H,H14), 2.87 (s, 6H, H(1, 2)), 2.02 (brs, 4H, H(15,20)), 1.89-1.80 (m, 2H,H21), 1.29 (t, J=7.06 Hz, 6H, H(24,25)); ¹³C NMR (100 MHz, CDCl₃, δ):151.79 (C3), 134.62 (C12), 129.79-128.60 (m, overlap, C5, C7, C9, C10,C11), 123.28 (C6), 119.28 (C8), 115.30 (C4), 62.23-62.16 (overlap, C16,C19, C22, C23), 51.31 (C17, C18), 45.43 (C1, C2), 39.73 (C14),24.75-22.83 (C15, C20, C21), 16.45 (²J=5.99, C24, C25) ppm; HRMS-DART(m/z): [M⁺] calculated for C₂₄H₄₁N₃O₅P₁S₁, 514.250. found, 514.251.

Example 233-(5-(dimethylamino)naphthalene-1-sulfonamido)-N,N-dimethyl-N-(3-phosphonopropyl)propan-1-ammoniumBromide (32)

Inside a flame dried and evacuated 20 mL screw cap vialN-(3-(diethoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide (0.35 g, 0.58 mmol) was dissolved in anhydrous DCM (5 mL). Tothe clear stirred solution was added TMSBr (0.23 mL, 1.76 mmol, 3.0 eq.)through a rubber septum via syringe and the reaction was stirred at roomtemperature overnight. Completion of the reaction was followed by ³¹Pafter which the reaction was quenched with EtOH (10 mL) and stirred for1 h followed by addition of H₂O (1 mL). Volatiles were removed with arotovap connected to a high vacuum Schlenk line and the crude productwas triturated with Et₂O (2×10 mL) to remove brown colored impurities.Further purification entailed extraction with NH₄ ⁺OH⁻:H₂O (1:10, 10 mL)and washing with Et₂O (1×5 mL). The aqueous fluorescent layer wasevaporated from ACN(1×50 mL) to give the pure product. Yield (0.25 g,79%). Light yellow solid. Mp=165-168° C.; ¹H NMR (400 MHz, MeOD, δ):8.52 (ddd, 1J=1.5 Hz, 2J=1.5 Hz, 3J=8.5 Hz, 1H, H9), 8.31 (ddd, ¹J=1.0Hz, ²J=1.0 Hz, ³J=8.5 Hz, 1H, H6), 8.23 (dd, ¹J=1.5 Hz, ²J=7.0 Hz, 1H,H11), 7.50-7.58 (m, 2H, H(5, 10)), 7.18 (dd, ¹J=1.0, ²J=7.5, 1H, H4),3.36-3.26 (s, m overlap, 6H, H(17, 18, 19)), 3.18-3.14 (m, 2H, H16),2.97 (t, J=6.02 Hz, 2H, H14), 2.89 (s, 6H, H(1, 2)), 1.93-1.86 (m, 4H,H(15,20)), 1.57-1.49 (m, 2H, H21), ppm; ¹³C NMR (100 MHz, MeOD, δ):133.80 (C3), 129.76-128.40 (m, overlap, C5, C7, C9, C10, C11, C12),124.54 (C6), 119.81 (C8), 116.59 (C4), 64.13 (C19), 61.33 (C16), 45.08(C17, C18), 34.04 (C1, C2), 24.77 (C14), 21.96 (C21), 16.85 (²J=3.24,C15) ppm; ³¹P NMR (121.45 MHz, MeOD, δ): 26.92 ppm; HRMS-DART (m/z):[M⁺] +calculated for C₂₃H₅₁NO₃P, 420.3601. found, 420.3608.

Synthesis of Bisphosphonic Acid Dansylphosphonic Acid Quats (BPDPQ)

Example 24N-(3-(diisopropoxyphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumBromide (33)

Diisopropyl (3-bromopropyl)phosphonate (0.964 g, 3.36 mmol) and DMOA (1g, 3.36 mmol, 1.0 eq.) were refluxed in ACN for 3 hrs. The mixture wasthen cooled to RT, poured into 20 mL of Et2O, and placed into a freezer(−20° C.) for 60 min to precipitate 33 as a white waxy solid. Yield: 89%(2.53 g). Mp=54-55° C.; ¹H NMR (400 MHz, CDCl3, δ): 4.68-4.60 (m, 2H,H10), 3.78-3.67 (m, 2H, H9) 3.50-3.42 (m, 2H, H8), 3.39 (s, 6H, H7),2.05-1.92 (m, 2H, H5), 1.83-1.62 (m, 4H, H4+H6), 1.28 (d, 2J=6.20 Hz,12H, H3) 1.21 (brs, 30H, H2), 0.84 (t, J=7.03 Hz, 3H, H1) ppm; ¹³C NMR(100 MHz, CDCl3, δ): 70.65 (d, 2J C-P=6.54 Hz, (C15)), 64.09 (C14),62.85 (d, 3J C-P=6.54 Hz, (C13)), 51.55 (C12), 31.84 (C11), 29.66-29.55(C10 overlap), 29.40 (C9), 29.29 (C8), 29.18 (C7), 22.41 (d, 2J C-P=Hz,(C6)), 23.95 (d, 1J C-P=Hz, (C5)), 22.61 (C4), 22.42 (C3), 16.70 (C2),14.06 (C1) ppm; ³¹P NMR (121.45 MHz, CDCl3, δ): 27.08 ppm. HRMS-DART(m/z): [M+] —Br— calculated for C₂₉H₆₃NO₃P, 504.4540. found, 504.4546.

Example 25 Applying Phosphonate Antimicrobial Coatings

The following general procedure may be used to treat a surface with aphosphonate antimicrobial coating:

1) Surface Pretreatment/Passivation modifies a metal surface by creatinga metal hydroxide layer to provide more binding sites on the surface ofthe material to which phosphonic acid compounds can bind can be achievedby mechanical means such as sanding, cleaning, or degreasing; chemicalmeans such as treatment with piranha solution (a 3:1 H₂SO₄/H₂O₂ forabout 10-30 minutes) or by activation by heating the surface (about 160°C. in air for about 1-2 hours).2) Coating application by dip coating at room temperature to about 50°C. and about 1 to about 10 mM solution of phosphonate/phosphonic acid inwater or alcohol; aerosol spraying an about 1 to about 10 mM solution ofphosphonate/phosphonic acid in water or alcohol solution; or by vapordeposition of volatile phosphonate/phosphonic acid on the passivatedsurface.3) Annealing to create strong molecule-material surface bonds by athermal cure at about 100 to about 140° C. in an oven, or preferably,120° C. for about 18 hours under about 0.1 Torr reduced pressure.4) Washing the treated surface to remove unbound phosphonate/phosphonicacid material via immersion/dipping with alcohol or water often with theuse of sonication.5) Repeating steps 2, 3 and 4 until such time as the surface issufficiently coated with the phosphonate antimicrobial coating

The following are examples of pretreatment and coating procedures for agiven substrate that may be applicable to the present invention:

Iron: grinding/polishing surface (600 grain size sand paper), dippinginto a solution of 10% HNO₃ (4 min at room temperature), followed bydegreasing in ethanol. Dip coating of phosphonate material (1 mM, 15hrs, water). (Hanson, E. L., Schwartz, J., Nickel, B., Koch, N. &Danisman, M. F. Bonding Self-Assembled, Compact OrganophosphonateMonolayers to the Native Oxide Surface of Silicon. J. Am. Chem. Soc.125, 16074-16080 (2003); Harm et al., Novel protective coating for steelbased on a combination of SAM and conducting polymers MacromolecularSymposia. 187, 65-72 (2002)) Novel protective coating for steel based ona combination of SAM and conducting polymers

Titanium foil: sanded, rinsed with hot methanol, and stored at 160° C.in air, gives a surface coating of hydroxylated titanium dioxide.Aerosol sprayed (0.75 mM in THF), annealed 18 hrs at 120° C., immersionin (dry THF twice, for 5 min each). {{5016 Gawalt, Ellen S. 2001;}}Similarly titanium disks were wet-ground (220-4000 grit silicon carbidepaper and further polished with OPChem polishing cloths using OP-Scolloidal silica suspension) followed by ultrasonication (deionizedwater to eliminate silica particles). Rinsed (acetone then ultrapurewater) and dried for a few minutes in an oven (80° C.). (Lecollinet, G.et al. Self-Assembled Monolayers of Bisphosphonates: Influence of SideChain Steric Hindrance. Langmuir 25, 7828-7835 (2009))

Stainless Steel: Mechanically Polished (220, 400, 800, and 1200 gritsilicon carbide paper followed by a 1 μm diamond suspension).Ultrasonicated (MeOH, 15 min) or (DCM (10 min) then acetone (10 min))and immersed (boiling MeOH to remove traces of organics and metallicdust), storage (120° C., oven). Dip coated (1 mM, dry tetrahydrofuran(THF)) and reduced pressure annealed (0.1 Torr). (Raman, A., Dubey, M.,Gouzman, I. & Gawalt, E. S. Formation of Self-Assembled Monolayers ofAlkylphosphonic Acid on the Native Oxide Surface of SS316L. Langmuir 22,6469-6472 (2006)) Similarly, oxidized SiO₂/Si, TiO₂/Ti and stainlesssteel samples were dip coated (10 min) wash cautiously with acetonefollowed by thermal annealing (24 hrs, 120° C.). Weakly adsorbedmolecules were removed from all coupons by 10 min sonication in acetone.Dipping, annealing, and sonicating steps were done twice. Water solublecoatings were dip coated (3 hours in water), no rinsing and annealed(120° C., 20 h) and sonicated for 10 min in ultrapure water. Dipping,drying, and sonicating were performed twice. (Lecollinet, G. et al.Self-Assembled Monolayers of Bisphosphonates: Influence of Side ChainSteric Hindrance. Langmuir 25, 7828-7835 (2009)).

Silicon (100) wafer: cleaning by sonication in acetone (15 min).oxidized (3:1, 30% H₂O₂: 98% H₂SO₄ for 30 min), and rinsed (ultrapurewater) and immediately dip coated (25 μM solution in THF until thesolvent evaporated at room temperature). Thermal annealed (140° C. 48hrs). Three cycles of depositions with multiple rinsing and sonicationin THF and methanol was used to produce a monolayer film. The films werestored in glass containers filled with nitrogen until they werecharacterized. (Hanson, E. L., Schwartz, J., Nickel, B., Koch, N. &Danisman, M. F. Bonding Self-Assembled, Compact OrganophosphonateMonolayers to the Native Oxide Surface of Silicon. J. Am. Chem. Soc.125, 16074-16080 (2003))

Non thermal annealing: titanium samples dip coated (1 mM solution inacetone, 3 hrs) decant solvent and reduced pressure anneal (15 h at 50°C.). The samples were ultrasonically washed with acetone and thenair-dried. (Lecollinet, G. et al. Self-Assembled Monolayers ofBisphosphonates: Influence of Side Chain Steric Hindrance. Langmuir 25,7828-7835 (2009))

Example 26 Minimum Inhibitory Concentration Determination

Solutions of phosphonate antimicrobials (0.01 g/mL or 1%) were dissolvedin H₂O or ethanol, inoculated with the test organism and seriallydiluted from 10⁻¹-10⁻³, representing a dilution range from 0.01 g/mL to0.00001 g/mL, followed by plate counts to determine the minimuminhibitory concentration (MIC) values. Test organisms includedgram-positive and gram-negative bacterial strains: Staphylococcusaureus, Listeria monocytogenes, Salmonella interiditis, and Escherichiacoli.

Sample 1 was a 1% solution of compound 3; sample 2 was 1% solution ofthe sodium salt of compound 3; sample 3 was a 1% solution of compound 3prepared by HCl dealkylation ofN-(3-diisopropoxyphosphorylpropyl)-N,N-dimethyloctadecan-1-ammoniumbromide.

Samples 1-3 prepared exhibited similar MIC values but demonstrated 100×higher efficacy with Listeria and Salmonella and 10× higher efficacyagainst E. coli compared toN-(3-trimethoxysilylpropyl)-N,N-dimethyloctadecan-1-ammonium chloride.All compounds were inhibitory towards the gram-positive S. aureusbacterium. A 1% solution of compound 2 was found to be highly inhibitoryagainst S. aureus even at 100 μg/mL concentrations.

A stock solution (1%) of theN-(3-trimethoxysilylpropyl)-N,N-dimethyloctadecan-1-ammonium chlorideprepared from the 5% stabilized solution in H₂O (stabilized) or preparedfrom the 72% concentrate in MeOH and diluted to 1% in H₂O (nostabilizer) worked extremely well. All bacterial strains tested withthese samples were inhibited at concentrations up 100 μg/mL which is inclose agreement with literature MIC values (84 μg/mL).

Example 27 Coating Composition of Compound-3-on TiO₂

A one inch by inch TiO₂ square was sanded with 600 grain size sandpaper, followed by an ethanol (EtOH) rinse. Samples were stored in a120° C. oven prior to use. A 10 mM solution of compound 3 in EtOH wasaerosol spayed onto the TiO₂ square, allowed to air dry and placed into120° C. oven overnight to anneal the compound followed by an EtOH rinse.Spaying, annealing and rinsing were repeated two more times.

Example 28 Contact Killing on Hard Surfaces

A common method to study long-term bacterial survival is using the largedroplet inoculation method, as it provides timely and reproducibleinoculation of a large number of test substrates and conditions (Jawad,A., Heritage, J., Snelling, A. M., Gascoyne-Binzi, D. M., & Hawkey, P.M. (1996). Influence of relative humidity and suspending menstrua onsurvival of Acinetobacter spp. on dry surfaces. Journal of ClinicalMicrobiology, 34(12), 2881-2887; Makison, C. & Swan, J. (2006). Theeffect of humidity on the survival of MRSA on hard surfaces. Indoor andBuilt Environment, 15(1), 85-91; Rose, L. J., Donlan, R., Banerjee, S.N., & Arduino, M. J. (2003) Survival of Yersinia pestis on environmentalsurfaces. Applied and Environmental Microbiology, 69(4), 2166-2171;Wendt, C., Dietz, B., Dietz, E., & Rüden, H. (1997). Survival ofAcinetobacter baumannii on dry surfaces. Journal of ClinicalMicrobiology, 35(6), 1394-1397; Yazgi, H., Uyanik, M. H., Ertek, M.,Akta

, A. E., Igan, H., & Ayyildiz, A. (2009). Survival of certain nosocomialinfectious agents on the surfaces of various covering materials. TurkishJournal of Medical Sciences, 39(4), 619-622).

The following bacteria were tested: Pseudomonas sp. CT07, Salmonellaenteriditis, Klebsiella pneumonia, Listeria monocytogenes, Arthrobacter,Staphylococcus aureus, Pseudomonas aeruginosa PA01 and Escherichia coli.The preparation of the inoculant was performed by growing overnightcultures (10⁵-10⁸ cfu/ml) in 10% tryptic soy broth at room temperatureand with agitation. The cells were then washed from the broth by using acentrifuge at 9000×g for 2 minutes. This step was performed to remove anutrition source for the bacteria during the test phase.

Stainless steel coupons were treated by overnight immersion in a 1%ethanolic solution (aqueous solutions may also be used) of antimicrobialphosphonate and an overnight cure at 100° C. Untreated control (“C”),compound 2 (“S1”), compound 3 (“S2”) and Bio-Protect® AM500 siliconequaternary ammonium salt(N-(3-(trimethoxy)silylpropyl)-N,N-dimethyloctadecan-1-ammoniumchloride) treated (“BSC”) stainless steel coupons were then inoculatedwith 0.1 mL of the overnight bacterial inoculant and were then placed ina fumehood to allow the samples to dry for 2 hours. After theappropriate time lapse the coupons were then placed into a 10 mL tube ofsterile 0.9% saline and vigorously vortexed for a minute to remove theadherent cells. A dilution series was made from this solution and theseries was plated onto 10% tryptic soy agar plates for enumeration. Thiswas again repeated after 4 hours, 6 hours and 24 hours of drying. Theresults are presented in FIGS. 1 and 2 demonstrating improvedantibacterial activity compared to control and commercially availableproduct-treated samples.

Titanium metal coupons were treated similarly as above and tested forantibacterial activity against Salmonella and S. aureus. Coupons treatedwith compound 2 obtained by dealkylation of compound 3 using TMSBr ordealkyation ofN-(3-(diisopropoxylphosphoryl)propyl)-N,N-dimethyloctadecan-1-ammoniumbromide (compound 33) using HCl showed significant reduction ofbacterial colonies after 3 hours of drying on the titanium surface.These results are presented in FIG. 3. Analogous tests using titaniumand stainless steel metal coupons against Arthrobacter showed similarantibacterial results. Analogous tests using titanium, stainless steeland aluminum metal coupons against P. aeroguinosa showed similarantibacterial results.

Example 29 Antimicrobial Surface Treatment Durability

Titanium metal coupons treated with compound 2 as in Example 28 werefurther tested for surface treatment durability. The treated couponswere stored in saline for 24 hours from the first antimicrobial trial,removed, dried, washed with distilled H₂O, dried re-innoculated withArthrobacter and retested. All samples showed similar colony reductions(10⁶ to 0) indicating the phosphonate molecule was truly immobilized.

The titanium coupon samples were subjected to agar testing in which thesamples were affixed to agar petri dishes and inoculated with P.aeroguinosa. While the control sample was fully colonized by bacteriathe samples treated with compound 2 had no visible colonies observedaround or on the samples.

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

We claim:
 1. An antimicrobial surface coating composition comprising acompound of formula (I)

wherein R₁ and R₂ are independently hydrogen, methyl, ethyl, isopropylor n-propyl; m is 15, 16, 17, 18 or 19; n is 0, 1, 2, 3, 4, 5 or 6; andX is chloro, bromo or iodo; and an environmentally friendly carrier. 2.The coating composition of claim 1 wherein R₁ and R₂ are methyl, m is17, n is 1 and X is bromo.
 3. The coating composition of claim 1 whereinthe carrier comprises a lower alkanol, water and mixtures thereof. 4.The coating composition of claim 3 wherein the lower alkanol is selectedfrom the group consisting of methanol, ethanol, n-propanol andi-propanol.